Formulations comprising anti-il-23p19 antibodies, methods of making and uses thereof

Abstract

The present invention relates to formulations comprising anti-IL-23p19 antibodies, in particular to pharmaceutical formulations comprising anti-IL-23p19 antibodies, a buffer, a stabilizer and a surfactant. Furthermore, the present invention relates to the disease-treating or -preventing use of these formulations.

Description

[0001] This application claims priority to Chinese patent application 2020104048344, filed on May 13, 2020. The entire contents of the aforementioned Chinese patent application are incorporated herein by reference. Technical Field

[0002] This invention relates to the field of antibody formulations. More specifically, this invention relates to pharmaceutical formulations comprising anti-IL-23p19 antibodies, particularly stable liquid formulations, lyophilized formulations, and reconstituted stable liquid formulations, as well as methods for preparing said pharmaceutical formulations, and the therapeutic and / or prophylactic uses of said pharmaceutical formulations. Background Technology

[0003] Interleukin (IL)-12 is a secreted heterodimeric cytokine composed of two disulfide-linked glycosylated protein subunits, named p35 and p40 due to their similar molecular weights. It has been discovered that the p40 protein subunit of IL-12 can also link with a separate protein subunit named p19 to form a new cytokine, interleukin-23 (IL-23).

[0004] Interleukin-23 (IL-23) is a heterodimeric cytokine comprising two subunits: p19 (I1-23p19), which is specific to IL-23, and p40 (IL-12p40), which is shared with IL-12. The p19 subunit is structurally related to IL-6, granulocyte colony-stimulating factor (G-CSF), and the p35 subunit of IL-12. IL-23 mediates signal transduction by binding to a heterodimeric receptor comprising two subunits: IL-23R, which is specific to the IL-23 receptor, and IL-12Rb1, which is shared with the IL-12 receptor.

[0005] Many early studies have demonstrated that the consequences of genetic defects in p40 (p40 knockout mice; p40KO mice) are more severe than those observed in p35-deficient mice (e.g., p35KO). These results are generally interpreted as p40 knockout preventing not only IL-12 expression but also IL-23 expression. See, for example, Oppmann et al. (2000) Immunity 13:715-725; Wiekowski et al. (2001) J. Immunol. 166:7563-7570; Parham et al. (2002) J. Immunol. 168:5699-708; Frucht (2002) Sci STKE 2002, E1-E3; Elkins et al. (2002) Infection Immunity 70:1936-1948). Recent studies have confirmed that IL-23 inhibition, achieved through neutralization in IL-23p19-deficient mice or with IL-23-specific antibodies, can provide benefits comparable to anti-IL-12p40 strategies (Cua et al., 2003; Murphy et al., 2003; Benson et al., 2004). Therefore, there is evidence of an increasing specific role of IL-23 in immune-mediated diseases. Neutralizing IL-23 does not inhibit the IL-12 pathway, thus providing effective treatment for immune-mediated diseases while having limited impact on important host defense mechanisms. This would represent a significant improvement over current treatment options. Therefore, there is a need in the art for novel IL-23p19 antibodies. Such IL-23p19 antibodies are described, for example, in patent application PCT / CN2019 / 121261.

[0006] Drug stability is a crucial indicator of drug efficacy and safety. Obtaining a suitable formulation is essential for maintaining the efficacy and safety of a drug throughout its shelf life. However, due to the complexity of antibodies themselves and their degradation pathways, it is currently impossible to predict the formulation conditions required for optimizing antibody stability. This is especially true considering that different antibodies often possess very different CDR sequences, and these sequence differences lead to varying stability properties in solution. Therefore, given the stringent requirements for the safety and efficacy of human antibodies, it is necessary to optimize the formulation individually for each antibody.

[0007] Although several IL-23p19 antibody formulations have been proposed, there remains a need in the field for novel pharmaceutical formulations containing sufficiently stable IL-23p19 suitable for administration to human subjects. Furthermore, for such antibody formulations, simplicity and ease of use in formulation formulation are also advantageous. Summary of the Invention

[0008] This invention addresses the aforementioned needs by providing a pharmaceutical formulation containing an antibody that specifically binds to IL-23p19. The antibody formulation of this invention exhibits excellent stability under varying temperature and time conditions.

[0009] In one aspect, the present invention therefore provides a liquid antibody formulation comprising (i) an anti-IL-23p19 antibody; (ii) a buffer; (iii) a stabilizer; and (iv) a surfactant.

[0010] In one embodiment, the anti-IL-23p19 antibody comprises a heavy chain variable region VH and a light chain variable region VL, wherein the heavy chain variable region comprises the sequence of SEQ ID NO: 7 or a sequence having at least 90% identity with it, and the light chain variable region comprises the sequence of SEQ ID NO: 8 or a sequence having at least 90% identity with it.

[0011] Sequence (SEQ ID NO: 7)

[0012]

[0013] Sequence (SEQ ID NO: 8)

[0014]

[0015] In one embodiment, the anti-IL-23p19 antibody comprises:

[0016] The heavy chain VH CDR1 of -GYTFTSYLMH (SEQ ID NO: 1);

[0017] The heavy chain VH CDR2 of -YINPYNEGTN (SEQ ID NO: 2);

[0018] The heavy chain VH CDR3 of -NWDLPY (SEQ ID NO: 3);

[0019] The light chain VL CDR1 of -RASQSISDYLH (SEQ ID NO: 4);

[0020] -YASQSMS (SEQ ID NO: 5) light chain VL CDR2; and

[0021] -QQGHSFPFT (SEQ ID NO: 6) light chain VL CDR3.

[0022] In one embodiment, the IL-23p19 antibody is an IgG1 type antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises the sequence of SEQ ID NO: 9 or a sequence having at least 90% identity with it, and wherein the light chain comprises the sequence of SEQ ID NO: 10 or a sequence having at least 90% identity with it.

[0023] Sequence (SEQ ID NO: 9)

[0024]

[0025]

[0026] Sequence (SEQ ID NO: 10)

[0027]

[0028] Preferably, the IL-23p19 antibody is the anti-IL-23p19 antibody 17D1-YTE disclosed in PCT application number PCT / CN2019 / 121261 (international application date: November 27, 2019), which consists of the heavy chain sequence of SEQ ID NO: 9 and the light chain sequence of SEQ ID NO: 10.

[0029] In one embodiment, the IL-23p19 antibody is recombinantly expressed in HEK 293 cells or CHO cells.

[0030] In one embodiment, the concentration of IL-23p19 antibody in the liquid antibody formulation of the present invention is about 1-300 mg / ml. In another embodiment, the concentration of IL-23p19 antibody in the liquid antibody formulation of the present invention is about 25-250 mg / ml, preferably 50-200 mg / ml, for example, concentrations of about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, and 200 mg / ml.

[0031] In one embodiment, the buffer in the liquid antibody formulation of the present invention is selected from histidine buffers, citrate buffers, acetate buffers, and phosphate buffers. Preferably, the buffer is selected from histidine, histidine hydrochloride, and combinations thereof. In one embodiment, the histidine buffer is selected from about 0.775-3.1 mg / mL histidine, preferably with a histidine concentration of about 1.55 mg / mL. In one embodiment, the histidine buffer is a combination of histidine and histidine hydrochloride, wherein the histidine content is about 0.38-1.52 mg / mL, and the histidine hydrochloride content is about 0.54-2.16 mg / mL, preferably with histidine and histidine hydrochloride concentrations of about 0.76 mg / mL and about 1.08 mg / mL, respectively.

[0032] In one embodiment, the liquid formulation of the present invention comprises sorbitol as a single stabilizer. In this embodiment, the amount of sorbitol in the liquid formulation of the present invention can be about 25-100 mg / ml, for example, 40-60 mg / ml. For example, sorbitol can be present in an amount of about 40, 42, 44, 46, 48, 50, 52, 54, 56, 58 or 60 mg / ml, preferably in an amount of about 50 mg / ml.

[0033] In one embodiment, the liquid formulation of the present invention comprises sucrose as a single stabilizer. In this embodiment, the amount of sucrose in the liquid formulation of the present invention can be about 40-160 mg / ml, preferably 70-90 mg / ml. For example, sucrose can be present in an amount of about 70, 75, 80, 85, or 90 mg / ml, preferably about 80 mg / ml. In one embodiment, the liquid formulation of the present invention comprises a combination of sorbitol and arginine as a stabilizer. In this combination, sorbitol can be present in an amount of about 15-60 mg / ml, preferably 20-40 mg / ml, for example, about 20, 25, 30, 35, or 40 mg / ml. In this combination, arginine can be present in an amount of about 6.97-27.88 mg / ml, preferably 10.45-17.42 mg / ml, particularly about 13.94 mg / ml. Preferably, the liquid formulation of the present invention comprises about 20-40 mg / ml of sorbitol and about 10.45-17.42 mg / ml of arginine. More preferably, the liquid formulation of the present invention comprises about 30 mg / ml of sorbitol and about 13.94 mg / ml of arginine.

[0034] In one embodiment, the liquid formulation of the present invention comprises a combination of sucrose and arginine as a stabilizer. In this combination, sucrose may be present at about 25-100 mg / ml, preferably 40-60 mg / ml, for example, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, or 60 mg / ml. In this combination, arginine may be present at an amount of about 6.97-27.88 mg / ml, preferably 10.45-17.42 mg / ml, and particularly about 13.94 mg / ml. Preferably, the liquid formulation of the present invention comprises about 40-60 mg / ml of sucrose and about 10.45-17.42 mg / ml of arginine. More preferably, the liquid formulation of the present invention comprises about 50 mg / ml of sucrose and about 13.94 mg / ml of arginine.

[0035] In one embodiment, the surfactant in the liquid antibody formulation of the present invention is a nonionic surfactant. In one embodiment, the surfactant is selected from polysorbate surfactants, poloxamer, and polyethylene glycol. In another embodiment, the surfactant is selected from polysorbate surfactants. In a specific embodiment, the surfactant in the liquid antibody formulation of the present invention is polysorbate-80 or polysorbate-20. In one embodiment, the concentration of the surfactant in the liquid antibody formulation of the present invention is about 0.1-1 mg / ml, preferably about 0.2-0.8 mg / ml, for example about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, or 0.8 mg / ml.

[0036] In one embodiment, the pH value of the liquid formulation is any value in the range of 5.2-6.3 (i.e., 5.5±0.3 to 6.0±0.3), for example, about 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, and 6.3. Preferably, the pH value of the formulation is 6.0±0.3.

[0037] In one embodiment, the liquid antibody formulation of the present invention comprises:

[0038] (i) Anti-IL-23p19 antibodies at concentrations of approximately 50-200 mg / ml, such as approximately 50, 100, 150, or 200 mg / ml;

[0039] (ii) Approximately 0.775–3.1 mg / mL of histidine;

[0040] (iii) Sorbitol at approximately 40-60 mg / mL; and

[0041] (iv) Polysorbate 80 at a concentration of approximately 0.2–0.8 mg / ml;

[0042] The pH of the liquid formulation is 6.0 ± 0.3, preferably 6.0;

[0043] Alternatively, the liquid antibody formulation contains

[0044] (i) Anti-IL-23p19 antibodies at concentrations of approximately 50-200 mg / ml, such as approximately 50, 100, 150, or 200 mg / ml;

[0045] (ii) Approximately 0.775–3.1 mg / mL of histidine;

[0046] (iii) Sorbitol at approximately 20-40 mg / mL, and arginine at approximately 10.45-17.42 mg / mL; and

[0047] (iv) Polysorbate 80 at a concentration of approximately 0.2–0.8 mg / ml;

[0048] The pH of the liquid formulation is 6.0 ± 0.3, preferably 6.0;

[0049] Alternatively, the liquid antibody formulation contains

[0050] (i) Anti-IL-23p19 antibodies at concentrations of approximately 50-200 mg / ml, such as approximately 50, 100, 150, or 200 mg / ml;

[0051] (ii) Approximately 0.775–3.1 mg / mL of histidine;

[0052] (iii) Approximately 40-60 mg / ml of sucrose, approximately 10.45-17.42 mg / ml of arginine; and

[0053] (iv) Polysorbate 80 at a concentration of approximately 0.2–0.8 mg / ml;

[0054] The pH of the liquid formulation is 6.0 ± 0.3, preferably 6.0;

[0055] Alternatively, the liquid antibody formulation contains

[0056] (i) Anti-IL-23p19 antibodies at concentrations of approximately 50-200 mg / ml, such as approximately 50, 100, 150, or 200 mg / ml;

[0057] (ii) Approximately 0.775–3.1 mg / mL of histidine;

[0058] (iii) Sucrose at approximately 70-90 mg / ml; and

[0059] (iv) Polysorbate 80 at a concentration of approximately 0.2–0.8 mg / ml;

[0060] The pH of the liquid formulation is 6.0 ± 0.3, preferably 6.0;

[0061] Alternatively, the liquid antibody formulation contains

[0062] (i) Anti-IL-23p19 antibodies at concentrations of approximately 50-200 mg / ml, such as approximately 50, 100, 150, or 200 mg / ml;

[0063] (ii) Approximately 0.38–1.52 mg / mL of histidine and approximately 0.54–2.16 mg / mL of histidine hydrochloride;

[0064] (iii) Sorbitol at approximately 40-60 mg / mL; and

[0065] (iv) Polysorbate 80 at a concentration of approximately 0.2–0.8 mg / ml;

[0066] The pH of the liquid formulation is 6.0 ± 0.3, preferably 6.0;

[0067] Alternatively, the liquid antibody formulation contains

[0068] (i) Anti-IL-23p19 antibodies at concentrations of approximately 50-200 mg / ml, such as approximately 50, 100, 150, or 200 mg / ml;

[0069] (ii) Approximately 0.38–1.52 mg / mL of histidine and approximately 0.54–2.16 mg / mL of histidine hydrochloride;

[0070] (iii) Sorbitol at approximately 20-40 mg / mL, and arginine at approximately 10.45-17.42 mg / mL; and

[0071] (iv) Polysorbate 80 at a concentration of approximately 0.2–0.8 mg / ml;

[0072] The pH of the liquid formulation is 6.0 ± 0.3, preferably 6.0;

[0073] Alternatively, the liquid antibody formulation contains

[0074] (i) Anti-IL-23p19 antibodies at concentrations of approximately 50-200 mg / ml, such as approximately 50, 100, 150, or 200 mg / ml;

[0075] (ii) Approximately 0.38–1.52 mg / mL of histidine and approximately 0.54–2.16 mg / mL of histidine hydrochloride;

[0076] (iii) Approximately 40-60 mg / ml of sucrose, approximately 10.45-17.42 mg / ml of arginine; and

[0077] (iv) Polysorbate 80 at a concentration of approximately 0.2–0.8 mg / ml;

[0078] The pH of the liquid formulation is 6.0 ± 0.3, preferably 6.0;

[0079] Alternatively, the liquid antibody formulation contains

[0080] (i) Anti-IL-23p19 antibodies at concentrations of approximately 50-200 mg / ml, such as approximately 50, 100, 150, or 200 mg / ml;

[0081] (ii) Approximately 0.38–1.52 mg / mL of histidine and approximately 0.54–2.16 mg / mL of histidine hydrochloride;

[0082] (iii) Sucrose at approximately 70-90 mg / ml; and

[0083] (iv) Polysorbate 80 at a concentration of approximately 0.2–0.8 mg / ml;

[0084] The pH of the liquid formulation is 6.0 ± 0.3, preferably 6.0;

[0085] The liquid formulation of the present invention can be stored stably for a long period of time, for example, at least 12 months or longer. In one embodiment, the liquid formulation of the present invention can be stored stably for at least 10 days, at least 20 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months, at least 36 months, or longer at about -80°C to about 45°C, for example, at -80°C, about -40°C, about -30°C, about -20°C, about 0°C, about 5°C, about 25°C, about 35°C, about 38°C, about 40°C, about 42°C, or about 45°C.

[0086] In one embodiment, the liquid formulation of the present invention can be stably stored for at least 12 months. In another embodiment, the liquid formulation of the present invention is stable at at least 40°C. In yet another embodiment, the liquid formulation of the present invention remains stable at about 2°C-8°C for at least 3 months, preferably at least 12 months, and more preferably at least 24 months. In one embodiment, the liquid formulation of the present invention remains stable at room temperature or, for example, about 25°C for at least 2 months, preferably at least 3 months, and more preferably at least 6 months.

[0087] In one embodiment, the stability of the formulation can be indicated by detecting changes in its appearance, visible foreign matter, protein content, turbidity, purity, and / or charge variants. In one embodiment, the stability of the liquid formulation of the present invention can be tested in a forced high-temperature stress test, such as after storage at 40°C ± 2°C for at least 1 week, 2 weeks, or preferably 1 month; or in an accelerated test, such as after storage at 25°C ± 2°C for at least 1 month or 2 months; or in a long-term test, such as after storage at 5°C ± 3°C for at least 2 months or 3 months. In one embodiment, the stability of the liquid formulation of the present invention is tested relative to an initial value, such as the initial value on day 0 of storage.

[0088] In one embodiment, after storage, the liquid formulation of the present invention remains clear to slightly opalescent in appearance, is a colorless to pale yellow liquid, and is free of foreign matter. In one embodiment, visual inspection under a clarity detector shows no visible foreign matter in the formulation. In one embodiment, after storage, the stability of the liquid formulation of the present invention is checked by measuring changes in protein content, wherein, for example by ultraviolet spectrophotometry (UV), the change in protein content relative to the initial value does not exceed 20%, preferably not more than 10%, for example 7-8%, more preferably not more than 5%, 2%, or 1%. In one embodiment, after storage, the stability of the liquid formulation of the present invention is checked by measuring changes in purity, wherein, by size exclusion high-performance liquid chromatography (SEC-HPLC), the change in monomer purity (or change in the main peak) relative to the initial value does not exceed 10%, for example not more than 5%, 4%, 3%, for example not more than 2%, preferably not more than 1%. In one embodiment, the stability of the liquid formulation of the present invention is examined after storage by measuring the change in purity, wherein the change in monomer purity (or change in the main peak) relative to the initial value is not more than 10%, for example, not more than 5%, 4%, 3%, 2%, or 1%, by non-reducing sodium dodecyl sulfate capillary electrophoresis (CE-SDS). In one embodiment, the stability of the liquid formulation of the present invention is detected after storage by cation exchange high-performance liquid chromatography (CEX-HPLC), wherein the percentage change in charge variants (e.g., the main component, acidic component, or basic component) in the formulation relative to the initial value is not more than 40%, for example, not more than 30% or not more than 20%; or the sum of the percentage changes in charge variants (the main component, acidic component, and basic component) is not more than 60%, for example, not more than 50% or not more than 40%.

[0089] In one embodiment, the quality standard for the actual production process is that the liquid formulation of the present invention is stable after storage, for example, after being stored at 5°C ± 3°C for at least 12 months, and preferably has one or more of the following characteristics: relative to the initial value on day 0 of storage,

[0090] (i) The formulation has a purity greater than 95%, preferably greater than 96%, 97%, 98%, or 99%, as measured by SEC-HPLC.

[0091] (ii) The formulation has a purity greater than 90%, preferably greater than 95%, 96%, 97%, or 98%, as measured by the non-reducing CE-SDS method;

[0092] (iii) The content of the main component in the formulation is greater than 50%, preferably greater than 60% or 70%, as measured by CEX-HPLC.

[0093] (iv) The protein content change rate measured by UV method is less than 10%, preferably less than 5%, 4%, 3%, or 2%.

[0094] In one aspect, the liquid formulation of the present invention is a pharmaceutical preparation, preferably an injection, more preferably a subcutaneous injection or an intravenous injection.

[0095] On the other hand, the present invention provides a solid antibody formulation obtained by solidifying the liquid antibody formulation of the present invention. The solidification process is carried out by, for example, crystallization, spray drying, or freeze-drying. In a preferred embodiment, the solid antibody formulation is, for example, a lyophilized powder for injection. The solid antibody formulation can be reconstituted in a suitable solvent before use to form the reconstituted formulation of the present invention. The reconstituted formulation is also a liquid antibody formulation of the present invention. In one embodiment, the suitable solvent is selected from water for injection, organic solvents for injection, including but not limited to oils for injection, ethanol, propylene glycol, etc., or combinations thereof.

[0096] In one aspect, the present invention provides a delivery device comprising the liquid or solid antibody formulation of the present invention. In one embodiment, the delivery device of the present invention is provided in the form of a pre-filled syringe comprising the liquid or solid antibody formulation of the present invention, for example for intravenous, subcutaneous, intradermal, or intramuscular injection, or intravenous infusion.

[0097] In another aspect, the present invention provides a method for delivering IL-23p19 antibody protein to a subject, such as a mammal, comprising the step of administering the subject a liquid or solid antibody formulation of the present invention, said delivery being carried out, for example, by using a delivery device using a pre-filled syringe.

[0098] In another aspect, the present invention provides the use of the liquid or solid antibody formulation of the present invention for the preparation of delivery devices or pre-filled syringes or drugs for treating immune system diseases, such as autoimmune diseases or inflammation, including (but not limited to) psoriasis, Crohn's disease, rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis, etc.

[0099] Other embodiments of the present invention will become clear from the following detailed description. Attached Figure Description

[0100] Combine with the following appendix Figure 1 Reading this description will provide a better understanding of the preferred embodiments of the invention as detailed below. For illustrative purposes, the figures show presently preferred embodiments. However, it should be understood that the invention is not limited to the precise arrangement and means of the embodiments shown in the figures.

[0101] Figure 1 pH screening test charge variants - acidic component variation graph (iCIEF method, 40℃±2℃).

[0102] Figure 2 pH screening test charge variants-principal component plot (iCIEF method, 40℃±2℃).

[0103] Figure 3 .Chart showing changes in charge variants and acidic components in the formulation determination experiment (CEX-HPLC method, 40℃±2℃).

[0104] Figure 4 Prescription determination experiment charge variant-principal component variation diagram (CEX-HPLC method, 40℃±2℃). Invention Details

[0106] Before describing the invention in detail, it should be understood that the invention is not limited to the specific methods and experimental conditions described herein, as these methods and conditions can be modified. Furthermore, the terminology used herein is for illustrative purposes only and is not intended to be restrictive.

[0107] definition

[0108] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For the purposes of this invention, the following terms are defined below.

[0109] The term “about” when used in conjunction with a numeric value means to cover a range of numeric values ​​that have a lower limit of 5% less than the specified numeric value and an upper limit of 5% greater than the specified numeric value.

[0110] When the term “and / or” is used to connect two or more options, it should be understood to mean any one of the options or any two or more of the options.

[0111] As used herein, the terms “comprising” or “including” mean to include the stated elements, integers, or steps, but do not exclude any other elements, integers, or steps. In this document, when the terms “comprising” or “including” are used, unless otherwise specified, they also cover situations consisting of the mentioned elements, integers, or steps. For example, when referring to an antibody variable region “comprising” a specific sequence, it is also intended to cover the antibody variable region consisting of that specific sequence.

[0112] The p19 subunit of IL-23 (also referred to herein as “IL-23p19” and “p19 subunit”) is a 189-amino acid polypeptide containing a 21-amino acid leader sequence (Oppmann et al., Immunity 13:715 (2000), SEQ ID NO: 181) and four packed α-helices, A, B, C, and D, in an up-up-down-down topology. The four helices are linked by three polypeptide loops. The AB and CD loops are made relatively long because they connect parallel helices. The short BC loop connects the antiparallel B and C helices. The p19 subunit of IL-23 is a member of the IL-6 family of helical cytokines. This cytokine family binds to its family-like receptors via three conserved epitopes (sites I, II, and III; Bravo and Heath (2000) EMBO J.19:2399-2411). The p19 subunit interacts with three cytokine receptor subunits to form a competent signal transduction complex. When expressed in cells, the p19 subunit first forms a complex with the p40 subunit, which is shared by IL-12. The p19p40 complex is secreted from the cell as a heterodimeric protein and is referred to as IL-23. In one embodiment, the IL-23p19 of the present invention is derived from human (NCBI: AAG37232) or cynomolgus monkey (NCBI: AEY84629).

[0113] As used herein, the terms “anti-IL-23p19 antibody,” “anti-IL-23p19,” “IL-23p19 antibody,” or “IL-23p19-binding antibody” refer to antibodies that are capable of binding with sufficient affinity to the (human or cynomolgus monkey) IL-23p19 subunit or fragment thereof such that the antibody can be used as a diagnostic and / or therapeutic agent targeting (human or cynomolgus monkey) IL-23p19.

[0114] In this article, the term “antibody” is used in the broadest sense to refer to a protein that contains an antigen-binding site, encompassing natural and artificial antibodies of various structures, including but not limited to complete antibodies and antigen-binding fragments of antibodies.

[0115] The terms “full-length antibody,” “complete antibody,” and “intact antibody” are used interchangeably herein to refer to a glycoprotein comprising at least two heavy chains (H) and two light chains (L) linked by disulfide bonds. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region. The heavy chain constant region consists of three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (VL) and a light chain constant region. The light chain constant region consists of one domain: CL. The VH and VL regions can be further subdivided into hypervariable regions (complementarity-determining regions (CDRs) interspersed with more conserved regions (framework regions (FRs)). Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The constant regions do not directly participate in antibody-antigen binding but exhibit various effector functions.

[0116] The complementarity-determining region (CDR) or CDR is a region within the antibody variable domain that is highly variable in sequence and forms a structurally defined loop ("hypervariant loop") and / or contains antigen contact residues ("antigen contact sites"). The CDR is primarily responsible for binding to antigen epitopes. The CDRs of the heavy and light chains are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus. CDRs located within the antibody heavy chain variable domain are referred to as HCDR1, HCDR2, and HCDR3, while those located within the antibody light chain variable domain are referred to as LCDR1, LCDR2, and LCDR3.

[0117] In a given light chain variable region or heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any of a number of known antibody CDR assignment systems or combinations thereof, including, for example: Chothia (Chothia et al., (1989) Nature 342: 877-883, Al-Lazikani et al., “Standard conformations for the canonical structures of immunoglobulins”, Journal of Molecular Biology, 273, 927-948 (1997)) based on antibody three-dimensional structure and CDR loop topology; Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th ed., USDapartment of Health and Human Services, National Institutes of Health (1987)) based on antibody sequence variability; AbM (University of Bath); Contact (University College London); and the International ImMunoGeneTics. The database (IMGT) (on the World Wide Web at imgt.cines.fr / ) and the North CDR definition based on affinity propagation clustering using a large number of crystal structures.

[0118] Unless otherwise stated, in this invention, when referring to the position of a residue in the antibody variable region (including heavy chain variable region residues and light chain variable region residues), it means the position numbered according to the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0119] In one embodiment, the CDR of the antibody of the present invention is defined by the AbM rule.

[0120] An "antibody fragment" refers to a molecule distinct from the intact antibody that contains a portion of the intact antibody and binds to the antigen bound by the intact antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; biantibodies; linear antibodies; single-chain antibodies (e.g., scFv); single-domain antibodies; bivalent or bispecific antibodies or fragments thereof; camelid antibodies; and bispecific or multispecific antibodies formed from antibody fragments.

[0121] The term "antibody preparation" refers to a preparation in a form that allows the biological activity of the antibody, as the active ingredient, to be effectively exerted, and which does not contain any other components that would have unacceptable toxicity to the subject to whom the preparation is administered. Such antibody preparations are typically sterile. Generally, antibody preparations contain pharmaceutically acceptable excipients. "Pharmaceutical-acceptable" excipients are reagents that can be rationally administered to test mammals so that an effective dose of the active ingredient used in the preparation can be delivered to the subject. The concentration of the excipient is adapted to the mode of administration, for example, it may be acceptable for injection.

[0122] The term "anti-IL-23p19 antibody formulation," also referred to herein as "the antibody formulation of the present invention," means a preparation comprising an anti-IL-23p19 antibody protein as the active ingredient and comprising a pharmaceutically acceptable excipient. When combined with a pharmaceutically acceptable excipient, the anti-IL-23p19 antibody protein as the active ingredient is suitable for therapeutic or prophylactic administration to humans or non-human animals. The antibody formulation of the present invention can be prepared, for example, as a liquid formulation in an aqueous form, such as for use with a pre-filled syringe, or as a lyophilized formulation, reconstituted (i.e., rehydration) by dissolving and / or suspending in a physiologically acceptable solution just before use. In some embodiments, the anti-IL-23p19 antibody protein formulation is in liquid form.

[0123] A "stable" antibody formulation means that the antibody in the formulation retains an acceptable level of physical and / or chemical stability after being stored under specific conditions, or after shaking, or after repeated freeze-thaw cycles. Although the antibody contained in the antibody formulation may not maintain 100% of its chemical structure after storage, shaking, or repeated freeze-thaw cycles, it is generally considered "stable" if it maintains about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% of the antibody structure or function. In some specific embodiments, the anti-IL-23p19 antibody protein formulation of the present invention exhibits low to undetectable antibody aggregation, degradation, or chemical modification during manufacturing, preparation, transportation, and long-term storage, resulting in minimal or even no loss of biological activity of the anti-IL-23p19 antibody protein, demonstrating high stability.

[0124] Various analytical techniques are known in the art for determining protein stability; see, for example, Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, NY, Pubs (1991) and Jones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993). Stability can be measured at selected temperatures and for selected storage times. For example, storage times can be selected based on the expected shelf life of the formulation. Alternatively, accelerated stability tests can be used. In some embodiments, stability testing is performed by subjecting the antibody formulation to various stress tests. These tests can represent extreme conditions that the formulated antibody formulation may encounter during manufacturing, storage, or transportation, or conditions that may accelerate the instability of the antibody in the formulation during non-manufacturing, storage, or transportation periods. For example, a formulated anti-IL-23p19 antibody protein formulation can be filled into glass vials to test antibody stability under high-temperature stress.

[0125] If, after a period of storage, the formulation does not exhibit aggregation, precipitation, turbidity, and / or denaturation; or exhibits very little aggregation, precipitation, turbidity, and / or denaturation, the antibody can be considered to have “maintained its physical stability” in the formulation. Since antibody aggregation in a formulation can potentially lead to an increased immune response in patients, resulting in safety concerns, it is necessary to minimize or prevent antibody aggregation in the formulation. SEC-HPLC can be used to determine soluble aggregates in the formulation. Furthermore, the stability of the formulation can be indicated by visually inspecting its appearance, color, and / or clarity; by detecting the turbidity of the formulation using the OD350nm method; or by determining the purity of the formulation using the non-reducing CE-SDS method. In one embodiment, the stability of the formulation is measured by determining the percentage of antibody monomers after storage at a specific temperature for a specific time, wherein a higher percentage of antibody monomers in the formulation indicates higher stability.

[0126] "Acceptable level" physical stability can be expressed as the presence of at least about 90% of the anti-IL-23p19 antibody protein monomers in the formulation after storage at a specific temperature for a specific period of time. In some embodiments, after storage at a specific temperature for at least 2 weeks, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months, or longer, acceptable level physical stability expresses at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the anti-IL-23p19 antibody protein monomers. When assessing physical stability, the specific storage temperature for pharmaceutical preparations can be any temperature from about -80°C to about 45°C, for example, stored at about -80°C, about -30°C, about -20°C, about 0°C, about 4°C-8°C, about 5°C, about 25°C, about 35°C, about 37°C, about 40°C, about 42°C, or about 45°C.

[0127] If the antibody in the formulation does not show significant chemical changes after a period of storage, it can be considered that the antibody "maintains its chemical stability" in the formulation. Most chemical instability stems from the formation of covalently modified forms of the antibody (e.g., charge variants). For example, basic variants can be formed by aspartic acid isomerization, N- and C-terminal modifications; acidic variants can be produced by deamidation, sialylation, and glycosylation. Chemical stability can be assessed by detecting and / or quantifying the chemically modified forms of the antibody. For example, charge variants of the antibody in the formulation can be detected by cation exchange chromatography (CEX) or imaging capillary isoelectric focusing electrophoresis (iCIEF). In one embodiment, the stability of the formulation is measured by determining the percentage change in the charge variant of the antibody in the formulation after storage at a specific temperature for a specific time, wherein a smaller change indicates higher stability of the formulation.

[0128] "Acceptable" chemical stability can be expressed as the percentage change in charge variants (e.g., the main component, acidic component, or basic component) in the formulation not exceeding 40%, for example, not exceeding 30% or 20%; or the sum of the percentage changes in charge variants (the main component, acidic component, and basic component) not exceeding 60%, for example, not exceeding 50% or 30%. In some embodiments, after storage at a specific temperature for at least 2 weeks, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months, or longer, acceptable chemical stability can be expressed as the percentage change in charge variants of the main component not exceeding about 50%, 40%, 30%, 20%, or 15%; or the sum of the percentage changes in charge variants not exceeding about 60%, 50%, or 30%. When assessing chemical stability, the storage temperature of the pharmaceutical preparation may be any temperature from about -80°C to about 45°C, for example, stored at about -80°C, about -30°C, about -20°C, about 0°C, about 4°C-8°C, about 5°C, about 25°C, or about 45°C.

[0129] The term "lyophilized formulation" refers to a composition obtained or that can be obtained by freeze-drying a liquid formulation. Preferably, it is a solid composition having a water content of less than 5%, preferably less than 3%.

[0130] The term "reconstituted formulation" refers to a liquid formulation obtained by dissolving and / or suspending a solid formulation (e.g., a lyophilized formulation) in a physiologically acceptable solution.

[0131] The term “room temperature” as used herein refers to a temperature of 15°C to 30°C, preferably 20°C to 27°C, and more preferably 25°C.

[0132] "Stress conditions" refer to chemically and / or physically unfavorable environments for antibody proteins, which can lead to unacceptable instability of the antibody protein, such as high temperature, agitation, or freeze-thaw cycles. "High-temperature stress" refers to storing the antibody preparation at room temperature or even higher temperatures (e.g., 40°C ± 2°C) for a period of time. Accelerated high-temperature stress tests can be used to examine the stability of antibody preparations.

[0133] As used herein, the term "parenteral administration" means administration other than enteral and local administration, typically by injection or infusion, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subcutaneous, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions. In some embodiments, the stable anti-IL-23p19 antibody formulation of the present invention is administered parenterally to a subject. In one embodiment, the anti-IL-23p19 antibody formulation of the present invention is administered to a subject by subcutaneous, intradermal, intramuscular, or intravenous injection.

[0134] I. Antibody preparations

[0135] This invention provides a stable liquid antibody formulation comprising (i) an anti-IL-23p19 antibody, (ii) a buffer, (iii) a stabilizer, and (iv) a surfactant, wherein the antibody formulation has a pH of about 5.2-6.3. In a preferred embodiment, the liquid antibody formulation of this invention is in injectable form.

[0136] (i) Anti-IL-23p19 antibody

[0137] In some embodiments, the anti-IL-23p19 antibody in the antibody formulation of the present invention comprises: a heavy chain variable region (VH) of SEQ ID NO: 7 or having at least 90% identity with it; and a light chain variable region (VL) of SEQ ID NO: 8 or having at least 90% identity with it.

[0138] In some embodiments, the anti-IL-23p19 antibody in the antibody formulation of the present invention comprises the VH CDR1, 2, and 3 sequences in the heavy chain variable region shown in SEQ ID NO: 7 and the VL CDR1, 2, and 3 sequences in the light chain variable region shown in SEQ ID NO: 8. In one embodiment, the anti-IL-23p19 antibody of the present invention has VH CDR1 of SEQ ID NO: 1, VH CDR2 of SEQ ID NO: 2, and VH CDR3 of SEQ ID NO: 3; and VL CDR1 of SEQ ID NO: 4, VL CDR2 of SEQ ID NO: 5, and VL CDR3 of SEQ ID NO: 6.

[0139] In some embodiments, the anti-IL-23p19 antibody in the antibody formulation of the present invention may comprise a heavy chain variable region (VH) having at least 90%, 95%, 98%, or 99% or higher identity with SEQ ID NO: 7; and / or a light chain variable region (VL) having at least 90%, 95%, 98%, or 99% or higher identity with SEQ ID NO: 8. In this document, “sequence identity” refers to the degree of sequence similarity on a nucleotide-by-nucleotide or amino acid-by-amino acid basis in a comparison window. The "sequence identity percentage" can be calculated as follows: Two best-aligned sequences are compared within a comparison window. The number of positions in the two sequences containing the same nucleic acid bases (e.g., A, T, C, G, I) or the same amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) is determined to obtain the number of matching positions. The number of matching positions is divided by the total number of positions in the comparison window (i.e., the window size), and the result is multiplied by 100 to produce the sequence identity percentage. The best alignment performed to determine the sequence identity percentage can be implemented in various ways known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (D NASTAR) software. Those skilled in the art can determine suitable parameters for aligning sequences, including any algorithms required to achieve maximum alignment across the full length of the sequence being compared or within the target sequence region.

[0140] In some embodiments, the VH sequence of the antibody of the present invention has no more than 10, preferably no more than 5, 4, or 3 different residues compared to SEQ ID NO: 7, and preferably the different residues are conserved amino acid substitutions. In some embodiments, the VL sequence of the antibody of the present invention has no more than 10, preferably no more than 5, 4, or 3 different residues compared to SEQ ID NO: 8, and preferably the different residues are conserved amino acid substitutions. "Conserved substitution" refers to an amino acid change that results in the replacement of a certain amino acid with a chemically similar amino acid. Providing a table of conserved substitutions of functionally similar amino acids is well known in the art.

[0141] In some embodiments, the anti-IL-23p19 antibody in the antibody formulation of the present invention is an IgG antibody. "IgG antibody" refers to an antibody whose heavy chain constant region belongs to the IgG form. All antibodies of the same IgG form have the same heavy chain constant region, while antibodies of different IgG forms have different heavy chain constant regions. For example, an IgG1 antibody refers to an antibody whose heavy chain constant region Ig domain is the Ig domain of IgG1.

[0142] In one embodiment of the present invention, the antibody described herein introduces substitution mutations (M252Y / S254T / T256E) in the Fc region to enhance its binding ability to human FcRn, thereby extending its half-life in vivo.

[0143] In a preferred embodiment, the anti-IL-23p19 antibody in the antibody formulation of the present invention is the anti-IL-23p19 antibody 17D1-YTE disclosed in PCT / CN2019 / 121261 (international application date: November 27, 2019), which has the heavy chain of SEQ ID NO: 9 and the light chain of SEQ ID NO: 10. In one embodiment, the anti-IL-23p19 antibody is an IgG1 type antibody produced by recombinant expression in CHO cells and then purified.

[0144] The amount of antibody or its antigen-binding fragment contained in the antibody formulation of the present invention may be varied depending on the specific purpose of the formulation, the specific environment, and the specific purpose for which the formulation is used. In some embodiments, the antibody formulation is a liquid formulation in which the concentration of IL-23p19 antibody is about 1-300 mg / ml. In another embodiment, the concentration of IL-23p19 antibody in the liquid antibody formulation of the present invention is about 25-250 mg / ml, preferably 50-200 mg / ml, for example, concentrations of about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 mg / ml.

[0145] (ii) Buffer

[0146] A buffer is a reagent that can maintain the pH of a solution within an acceptable range. In some embodiments, the buffer used in the formulation of the present invention can control the pH of the formulation of the present invention within a pH range of approximately 5.0-6.0, for example, approximately 5.5-6.0. In some specific embodiments, the antibody formulation of the present invention has a pH of approximately 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, or 6.3. For example, the antibody formulation of the present invention has a pH of 5.5 ± 0.3 or 6.0 ± 0.3, preferably a pH of 6.0.

[0147] In some embodiments, the formulation of the present invention comprises a buffer system selected from the following: histidine-histidine hydrochloride buffer system, citrate-sodium citrate buffer system, acetate-sodium acetate buffer system, phosphate buffer system, preferably histidine-histidine hydrochloride buffer system.

[0148] In some embodiments, the buffer used in the formulation of the present invention is selected from histidine, histidine hydrochloride, and combinations thereof. In some embodiments, the concentration of histidine in the buffer of the present invention is about 0.775-3.1 mg / mL histidine, preferably about 1.55 mg / mL histidine. In another embodiment, the buffer used in the formulation of the present invention is composed of histidine and histidine hydrochloride, wherein the histidine content is about 0.38-1.52 mg / mL and the histidine hydrochloride content is about 0.54-2.16 mg / mL, preferably the concentrations of histidine and histidine hydrochloride are about 0.76 mg / mL and about 1.08 mg / mL, respectively.

[0149] (iii) Stabilizer

[0150] Suitable stabilizers for use in this invention may be selected from sugars, polyols, and amino acids, and combinations thereof. Sugars used as stabilizers include, but are not limited to, sucrose, trehalose, maltose, and combinations thereof. Polyols used as stabilizers include, but are not limited to, sorbitol, mannitol, or combinations thereof. Amino acids used as stabilizers include, but are not limited to, arginine, arginine hydrochloride, methionine, glycine, proline, and combinations thereof.

[0151] For example, in some embodiments, the stabilizer comprises one or more of the following:

[0152] In one embodiment, the liquid formulation of the present invention comprises sorbitol as a single stabilizer. In this embodiment, the amount of sorbitol in the liquid formulation of the present invention can be about 25-100 mg / ml, for example, 40-60 mg / ml. For example, sorbitol can be present in an amount of about 40, 42, 44, 46, 48, 50, 52, 54, 56, 58 or 60 mg / ml, preferably in an amount of about 50 mg / ml.

[0153] In one embodiment, the liquid formulation of the present invention comprises a combination of sorbitol and arginine as a stabilizer. In this combination, sorbitol may be present at about 15-60 mg / ml, preferably 20-40 mg / ml, for example, about 20, 25, 30, 35, or 40 mg / ml. In this combination, arginine may be present at an amount of about 6.97-27.88 mg / ml, preferably 10.45-17.42 mg / ml, particularly about 13.94 mg / ml. Preferably, the liquid formulation of the present invention comprises about 20-40 mg / ml of sorbitol and about 10.45-17.42 mg / ml of arginine. More preferably, the liquid formulation of the present invention comprises about 30 mg / ml of sorbitol and about 13.94 mg / ml of arginine.

[0154] In one embodiment, the liquid formulation of the present invention comprises sucrose as a single stabilizer. In this embodiment, the amount of sucrose in the liquid formulation of the present invention may be about 40-160 mg / ml, preferably 70-90 mg / ml. For example, sucrose may be present in an amount of about 70, 75, 80, 85 or 90 mg / ml, preferably in an amount of about 80 mg / ml.

[0155] In one embodiment, the liquid formulation of the present invention comprises a combination of sucrose and arginine as a stabilizer. In this combination, sucrose may be present at about 25-100 mg / ml, preferably 40-60 mg / ml, for example, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, or 60 mg / ml. In this combination, arginine may be present at an amount of about 6.97-27.88 mg / ml, preferably 10.45-17.42 mg / ml, and particularly about 13.94 mg / ml. Preferably, the liquid formulation of the present invention comprises about 40-60 mg / ml of sucrose and about 10.45-17.42 mg / ml of arginine. More preferably, the liquid formulation of the present invention comprises about 50 mg / ml of sucrose and about 13.94 mg / ml of arginine.

[0156] (iv) Surfactants

[0157] As used herein, the term "surfactant" refers to an organic substance with an amphiphilic structure; that is, it consists of groups with opposite solubility tendencies, typically oil-soluble hydrocarbon chains and water-soluble ionic groups.

[0158] In one embodiment, the surfactant in the liquid formulation of the present invention is a nonionic surfactant, such as an alkyl poly(ethylene oxide). Specific nonionic surfactants that may be included in the formulation of the present invention include, for example, polysorbates, such as polysorbate-20, polysorbate-80, polysorbate-60, or polysorbate-40; poloxamer, etc. In a preferred embodiment, the liquid formulation of the present invention contains polysorbate-80 or polysorbate-20 as a surfactant.

[0159] The amount of surfactant contained in the antibody formulation of the present invention can be varied depending on the specific target characteristics of the formulation, the specific environment, and the specific purpose for which the formulation is used. In some preferred embodiments, the formulation may contain about 0.1-1 mg / ml, preferably about 0.2-0.8 mg / ml, for example about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, or 0.8 mg / ml of surfactant, especially polysorbate-80, preferably about 0.5 mg / ml of polysorbate-80.

[0160] (v) Other excipients

[0161] The antibody liquid formulations of the present invention optionally include other excipients. These other excipients include, for example, antimicrobial agents, antistatic agents, antioxidants, chelating agents, gelatin, etc. These and other known pharmaceutical excipients and / or additives suitable for the formulations of the present invention are well known in the art, for example, as listed in "The Handbook of Pharmaceutical Excipients, 4th Edition, edited by Rowe et al., American Pharmaceuticals Association (2003); and Remington: the Science and Practice of Pharmacy, 21st Edition, edited by Gennaro, Lippincott Williams & Wilkins (2005)".

[0162] II. Preparation of the formulation

[0163] This invention provides a stable formulation comprising an anti-IL-23p19 antibody protein. The anti-IL-23p19 antibody protein used in the formulation of this invention can be prepared using techniques known in the art for antibody production. For example, the antibody can be prepared recombinantly. In a preferred embodiment, the antibody of this invention is prepared recombinantly in 293 cells or CHO cells.

[0164] Antibodies are now widely used as active pharmaceutical ingredients. Techniques for purifying therapeutic antibodies to pharmaceutical grade are well-known in the field. For example, Tugcu et al. (Maximizing productivity of chromatography steps for purification of monoclonal antibodies, Biotechnology and Bioengineering 99 (2008) 599-613.) describe a three-column purification method for monoclonal antibodies using ion exchange chromatography (anion IEX and / or cation CEX chromatography) after a protein A capture step. Kelley et al. (Weak partitioning chromatography for anion exchange purification of monoclonal antibodies, Biotechnology and Bioengineering 101 (2008) 553-566) describe a two-column purification method using a weakly partitioning anion exchange resin after protein A affinity chromatography.

[0165] Generally, recombinant monoclonal antibodies can be purified using conventional purification methods to provide pharmaceutical substances with sufficient reproducibility and adequate purity for the formulation of antibody preparations. For example, after the antibody is secreted from the recombinant expression cells into the culture medium, the supernatant from the expression system can be concentrated using a commercially available protein concentrator filter, such as an Amicon ultrafiltration device. The antibody can then be purified using methods such as chromatography, dialysis, and affinity purification. Protein A is well-suited as an affinity ligand for the purification of IgG1, IgG2, and IgG4 type antibodies. Other antibody purification methods, such as ion exchange chromatography, can also be used. After obtaining antibodies of sufficient purity, formulations containing the antibodies can be prepared according to methods known in the art.

[0166] For example, the preparation can be carried out using the following steps: (1) after fermentation, centrifuge the fermentation broth to clarify and remove impurities such as cells to obtain a supernatant; (2) capture the antibody using affinity chromatography (e.g., a protein A column with specific affinity for IgG1, IgG2, and IgG4 antibodies); (3) inactivate the virus; (4) purify the protein (usually using CEX cation exchange chromatography) to remove impurities from the protein; (5) filter the virus (to reduce the virus titer by, for example, more than 4 log10); (6) ultrafiltration / distillation (which can be used to replace the protein with a formulation buffer that is conducive to its stability and concentrate it to a suitable concentration for injection). See, for example, B. Minow, P. Rogge, K. Thompson, BioProcess International, Vol. 10, No. 6, 2012, pp. 48-57.

[0167] III. Analytical methods for formulations

[0168] Stability studies of biological products generally include real-time stability studies (long-term stability studies) under actual storage conditions, accelerated stability studies, and forced-condition testing studies. Stability studies should explore and optimize research conditions based on the research objectives and the product's characteristics; long-term, accelerated, and / or forced-condition testing protocols should be developed to address various influencing factors. Accelerated and forced-condition testing are helpful in understanding the product's stability under short-term deviations from storage conditions and extreme conditions, and provide supporting data for determining shelf life and storage conditions.

[0169] During the storage of antibody preparations, antibodies may aggregate, degrade, or undergo chemical modification, leading to antibody heterogeneity (including size and charge heterogeneity) and the formation of aggregates and fragments, thereby affecting the quality of the antibody preparations. Therefore, it is necessary to monitor the stability of antibody preparations.

[0170] Various methods are known in the art for detecting the stability of antibody formulations. For example, the purity of antibody formulations and the level of antibody aggregation can be analyzed and assessed using methods such as reduced CE-SDS, non-reduced CE-SDS, and SEC-HPLC; charge variants in antibody formulations can be analyzed using methods such as cIEF, iCIEF, and ion exchange chromatography (IEX). Furthermore, the stability of a formulation can be rapidly determined by visually inspecting its appearance. Additionally, changes in protein content in the formulation can be detected using ultraviolet spectrophotometry (UV method).

[0171] Non-reducing CE-SDS is a capillary-based method for determining antibody purity. In CE-SDS, protein migration is driven by the surface charge induced by SDS binding, which is proportional to the protein's molecular weight. Since all SDS-protein complexes have similar mass-to-charge ratios, electrophoretic separation based on molecular size or hydrodynamic radius can be achieved in a capillary molecular sieve gel matrix. This method has been widely used to monitor the purity of denatured, intact antibodies. Generally, in non-reducing CE-SDS, the test sample is mixed with SDS sample buffer and iodoacetamide. The mixture is then incubated at 68-72°C for approximately 10-15 minutes, cooled to room temperature, and centrifuged. The supernatant is used for analysis. Protein migration is detected using a UV detector to obtain an electrophoretic pattern. Antibody purity can be calculated as the percentage of the IgG main peak area relative to the sum of all peak areas. For a further description of the CE-SDS method, please refer to, for example, Richard R. et al., Application of CE SDS gel in development of biopharmaceutical antibody-based products, Electrophoresis, 2008, 29, 3612-3620.

[0172] Size exclusion high-performance liquid chromatography (SEC-HPLC) is another important method for antibody standardization and quality control. This method primarily separates molecules based on differences in molecular size or hydrodynamic radius. SEC-HPLC can separate antibodies into three main forms: high molecular weight monosodium molecule (HMMS), the main peak (primarily antibody monomer), and low molecular weight monosodium molecule (LMMS). Antibody purity can be calculated as the percentage of the main peak area relative to the sum of all peak areas on the chromatogram. SEC-HPLC can also measure the percentage of antibody monomers in a pharmaceutical product, providing information on the content of soluble aggregates and shear products. For further description of the SEC-HPLC method, see, for example, J. Pharm. Scien., 83: 1645-1650, (1994); Pharm. Res., 11: 485 (1994); J. Pharm. Bio. Anal., 15: 1928 (1997); J. Pharm. Bio. Anal., 14: 1133-1140 (1986). Also see, for example, R. Yang et al., High-resolution separation of recombinant monoclonal antibodies by size exclusion ultra-high performance liquid chromatography (SE-UHPLC), Journal of Pharmaceutical and Biomedical Analysis (2015). http: / / dx.doi.org / 10.1016 / j.jpba.2015.02.032 ; and Alexandre Goyon et al., Protocols for the analytical characterization of therapeutic monoclonal antibodies. I-Non-denaturingchromatographic techniques, Journal of Chromatography, http: / / dx.doi.org / 10.1016 / j.jchromb.2017.05.010 .

[0173] Charge variants of antibodies in antibody preparations can be determined by cation exchange high-performance liquid chromatography (CEX-HPLC). In this assay, peaks eluting from the CEX-HPLC column earlier than the retention time of the main peak (or main component) are labeled as "acidic peaks" (or acidic components), while peaks eluting from the CEX-HPLC column later than the retention time of the main peak are labeled as "basic peaks" (or basic components).

[0174] Accelerated stability studies can be used to examine the stability properties of a product, which is beneficial for screening stable drug formulations. For example, formulation samples can be placed at elevated temperatures, such as approximately 40°C ± 2°C or 25°C ± 2°C, to conduct accelerated stability studies. Indicators for product stability can include appearance, visible foreign matter, protein content, turbidity, purity (SEC-HPLC, non-reducing CE-SDS), and charge variants (jCIEF, CEX-HPLC).

[0175] IV. Uses of the formulation

[0176] This invention provides formulations for treating IL-23-related diseases in subjects. Subjects may be mammals, such as primates, preferably higher primates, such as humans (e.g., patients with or at risk of having the diseases described herein). In one embodiment, the subject has or is at risk of having a disease described herein (e.g., an IL-23-related disease as described herein, such as an immune system disease (e.g., an autoimmune disease or inflammation)). In some embodiments, the subject has received or has received other treatments, such as anti-inflammatory or immunosuppressant therapy and / or radiation therapy.

[0177] In some implementations, the IL-23-related diseases described herein include immune system diseases, such as autoimmune diseases and inflammatory diseases. These diseases include (but are not limited to) psoriasis, Crohn's disease, rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis, etc.

[0178] In one implementation, an immune system disease is a disease that expresses elevated levels of IL-23p19.

[0179] In some embodiments, the preventive or treatment methods described herein also include administering, in combination to the subject or individual, the antibody molecules or pharmaceutical compositions or immunoconjugates disclosed herein, and one or more other therapies, such as modes of treatment and / or other therapeutic agents.

[0180] The present invention also provides the use of the formulations of the present invention in the preparation of a medicament for delivering an anti-IL-23p19 antibody protein to a mammal.

[0181] The antibody preparation of the present invention can be administered to subjects or patients via a variety of routes. For example, administration can be performed by infusion or via syringe. Therefore, in one aspect, the present invention provides a delivery device (e.g., a syringe) comprising the antibody preparation of the present invention (e.g., a pre-filled syringe). The patient will receive an effective amount of anti-IL-23p19 antibody protein as the main active ingredient, i.e., an amount sufficient to treat, improve, or prevent a target disease or condition.

[0182] Therapeutic effects may include reduction of physiological symptoms. The optimal effective dose and concentration of an antibody for any particular subject will depend on a variety of factors, including the patient's age, weight, health status and / or sex, the nature and extent of the disease, the activity of the specific antibody, the body's clearance of it, and any other possible treatments administered in combination with the antibody preparation. For specific cases, the effective dose delivered can be determined within the judgment of the clinician.

[0183] The following embodiments are described to aid in understanding the invention. It is not intended, and should not be construed in any way, as limiting the scope of the invention.

[0184] Abbreviation Description

[0185] abbreviations Full name CE-SDS Sodium dodecyl sulfate capillary gel electrophoresis CEX-HPLC Cation exchange high performance liquid chromatography ELISA Enzyme-linked immunosorbent assay SEC-HPLC Size exclusion high performance liquid chromatography iCIEF Imaging capillary isoelectric focusing electrophoresis Detailed Implementation

[0186] The anti-IL-23p19 antibody 17D1-YTE of this invention is an antibody independently developed by Innovent Biologics (Suzhou) Co., Ltd., and is disclosed in PCT application number PCT / CN2019 / 121261.

[0187] To develop an injectable formulation suitable for long-term stable storage and easy use of the antibody of this invention, the effects of different pH values ​​and the content of different stabilizers on the protein content of the antibody were investigated through forced stability experiments at 40°C and accelerated stability experiments at 25°C. Finally, a formulation favorable for its stability was selected. The materials and methods used in the entire research process are as follows:

[0188] Materials and methods

[0189] 1.1. Materials used in the formulation research of this invention

[0190]

[0191]

[0192] Note: N / A indicates not applicable.

[0193] 1.2. Instruments and equipment used in the formulation research of this invention

[0194] name Origin and Brand model serial number Electronic balance Sartorius, Germany BSA3202S PD-A1-186 Constant temperature and humidity chamber BINDER, Germany KBF P 720 PD-A1-070 Biochemical incubator Shanghai Jinghong SHP-150 PD-A1-200 Medical refrigerator Qingdao Haier HYC-360 PD-A1-165 Ultra-low temperature freezer Thermo USA 907 PD-A1-175 Clarity tester Tianjin Tianda Tianfa YB-2 PD-A1-033 UV-Vis spectrophotometer Shimadzu UV-1800 AS-A1-037 pH meter Mettler Switzerland FE20 PD-A1-161 Multichannel micro spectrophotometer Thermo USA Nanodrop 8000 PD-A1-052 benchtop refrigerated centrifuge Thermo USA SL16R PD-A1-082 Clean bench Suzhou Airtech SW-CJ-2FD QC-A1-011 Medium flow manual peristaltic pump Watson Marlow, UK 520S / R2 PD-A1-235 Filling machine Watson Marlow, Denmark FP50 PD-C14-115 Insoluble particulate detector Tianjin Tianda Tianfa GWJ-8 QC-A1-094

[0195] 1.3. Test items and methods for formulation stability

[0196] The main testing items during the entire research process included: (1) detecting the appearance and presence of visible foreign matter; (2) determining the protein content in the preparation by ultraviolet (UV) method; (3) determining the purity of the antibody preparation by size exclusion high performance liquid chromatography (SEC-HPLC), expressed as the percentage of the area of ​​the main peak to the sum of the areas of all peaks; (4) determining the purity of the antibody preparation by non-reduced sodium dodecyl sulfate capillary electrophoresis (non-reduced CE-SDS), expressed as the percentage of the area of ​​the main peak to the sum of the areas of all peaks; (5) determining the charge variants in the antibody preparation by CEX-HPLC method, expressed as the percentage of the main component, acidic component and basic component; (6) determining the charge variants in the antibody preparation by iCIEF method, expressed as the percentage of the main component, acidic component and basic component.

[0197] Visible foreign matter inspection and insoluble particulate matter detection

[0198] According to the methods described in the Pharmacopoeia of the People's Republic of China, a clarity analyzer (manufactured by Tianjin Tianda Tianfa, model YB-2) was used to check for visible foreign matter in the samples. An insoluble particulate matter analyzer (manufactured by Tianjin Tianda Tianfa, model GWJ-8) was used to check for insoluble particulate matter in the samples.

[0199] Protein content determination

[0200] The protein content in the sample was determined using a UV spectrophotometer (Shimadzu UV-1800, Japan) or a multi-channel micro spectrophotometer (Thermo Nanodrop 8000, USA).

[0201] Purity (SEC-HPLC method)

[0202] Separation was performed using a size exclusion column. The mobile phase was phosphate buffer (3.12 g sodium dihydrogen phosphate dihydrate, 8.77 g sodium chloride, and 34.84 g arginine were dissolved in ultrapure water, and the pH was adjusted to 6.8 with hydrochloric acid and brought to a final volume of 1000 ml). The column protectant was 0.05% (w / v) NaN3. The injection volume was 50 μl, the flow rate was 0.5 ml / min, the acquisition time was 30 min, the column temperature was 25℃, and the detection wavelength was 280 nm. The test sample was diluted with ultrapure water to a concentration of 2 mg / ml to prepare the test solution. The formulation buffer was diluted using the same method to prepare the blank solution. 50 μl each of the blank solution and the test solution were injected into the liquid chromatograph, and detection was initiated.

[0203] Purity (non-reducing CE-SDS method)

[0204] Capillary gel electrophoresis was used for detection. The capillary was an uncoated capillary with an inner diameter of 50 μm, a total length of 30.2 cm, and an effective length of 20.2 cm. Before electrophoresis, the capillary column was rinsed with 0.1 mol / L sodium hydroxide, 0.1 mol / L hydrochloric acid, ultrapure water, and electrophoresis gel at 70 psi. Dilute the sample to be tested with an appropriate amount of ultrapure water to 2.0 mg / mL. Take 50 μl of the diluted sample into a 1.5 mL centrifuge tube and add 45 μl of pH 6.5 sample buffer (weigh 0.32 g of citrate monohydrate and 2.45 g of disodium hydrogen phosphate dodecahydrate, dissolve in 45 mL of ultrapure water, and bring the volume to 50 mL to prepare citrate-phosphate buffer; accurately measure 200 μl of this buffer, add 80 μl of 10% (w / v) sodium dodecyl sulfate solution, add water to 1 mL, and mix well), 1 μl of internal standard (10 kDa protein, 5 mg / mL) (Beckman Coulter, catalog number: 390953), and 5 μl of... A 250 mmol / L NEM solution (62 mg of N-ethylcis-butyrendiimide dissolved in 2 ml of ultrapure water) was prepared, thoroughly mixed, heated at 70 ± 2 °C for 10 ± 2 minutes, cooled to room temperature, and transferred to a sample vial as the test solution. A blank solution was prepared by taking the same volume of the formulation buffer as the test solution and performing the same procedure. Sample injection conditions: -5 kV for 20 seconds; separation voltage: -15 kV for 35 minutes. The capillary column temperature was controlled at 25 °C, and the detection wavelength was 220 nm.

[0205] Charge variants (CEX-HPLC method)

[0206] The sample was detected using cation exchange chromatography (CEX-HPLC). A MabPac SCX-10 strong cation exchange column was used for separation. Mobile phase A was 10 mmol / L phosphate buffer (1.33 g of NaH₂PO₄·2H₂O and 0.54 g of Na₂HPO₄·12H₂O were dissolved in 800 mL of ultrapure water and brought to a final volume of 1000 mL, then filtered through a 0.22 μm filter). Mobile phase B was 10 mmol / L phosphate + 500 mmol / L sodium chloride buffer (1.33 g of NaH₂PO₄·2H₂O, 0.54 g of Na₂HPO₄·12H₂O and 29.22 g of NaCl were dissolved in 800 mL of ultrapure water and brought to a final volume of 1000 mL, then filtered through a 0.22 μm filter). The sample was diluted to 2.0 mg / mL with ultrapure water to prepare the test solution. Dilute the formulation buffer solution using the same method described above to prepare a blank solution. Inject 50 μl each of the blank solution and the test solution into the liquid chromatograph. Set the mobile phase flow rate to 1.0 mL / min, acquisition time to 35 minutes, column temperature to 35°C, detection wavelength to 280 nm, and sample pan temperature to 10°C. Perform injection analysis and calculate the contents of the principal component, acidic component, and basic component using the area normalization method.

[0207] Charge variant (iCIEF method)

[0208] Imaging capillary isoelectric focusing electrophoresis (iCIEF) was used for detection. The capillary had an inner diameter of 100 μm and a total length of 5 cm. Before electrophoresis, the capillary column was rinsed with 0.5% methylcellulose solution (hereinafter referred to as MC solution) and ultrapure water. Vacuum injection was used for 55 seconds, with a pre-focusing voltage and time of 1.5 kV for 1 minute, a focusing voltage and time of 3 kV for 8 minutes, an injection time of 55 seconds, a sample pan temperature of 10℃, a capillary column temperature of room temperature, and a detection wavelength of 280 nm. The cathodic stabilizer consisted of a 500 mmol / L arginine solution, 3 mol / L urea to improve protein solubility, and 0.5% MC solution to reduce protein adhesion to the capillary. The test sample was diluted with water to 1 mg / ml. 20 μl of the diluted test sample solution was added to 78 μl of premixed solution and thoroughly mixed to prepare the test sample solution. A blank solution was prepared using the same procedure with the formulation buffer.

[0209] Example

[0210] Example 1. Preparation and purification of IL-23p19 antibody

[0211] As described in PCT application number PCT / CN2019 / 121261, the antibody 17D1-YTE that specifically binds to IL-23p19 was obtained. This antibody has a heavy chain sequence of SEQ ID NO: 9 and a light chain sequence of SEQ ID NO: 10. The PCT application number PCT / CN2019 / 121261 is hereby incorporated herein by reference in its entirety.

[0212] Briefly, the antibody was recombinantly expressed in CHO cells, and the IL-23p19 antibody sample used in the pH screening experiment of the present invention was purified by affinity chromatography, and the IL-23p19 antibody sample used in the formulation screening experiment of the present invention was purified by cation exchange chromatography.

[0213] Example 2. pH Screening Test

[0214] 2.1 Experimental Procedure

[0215] In this example, the effects of citrate buffer systems at pH 5.0, 5.5, 6.0, and 6.5 on the stability of the purified IL-23p19 antibody of Example 1 were investigated to obtain a better pH value range.

[0216] Prepare 20 mM sodium citrate and 150 mM sodium chloride, and adjust the pH to 5.0, 5.5, 6.0, and 6.5 respectively with hydrochloric acid. Ultrafilter and replace the purified IL-23p19 antibody of Example 1 into the above buffers with different pH values, and adjust the protein content to 50 mg / ml; filter and dispense into vials, stopper, and crimp the caps. Place the above samples in a constant temperature and humidity incubator at 40°C ± 2°C, take samples at 0, 1, 3, 5, and 10 days, and store them in a cryogenic refrigerator after sampling. After thawing and mixing evenly, send them for inspection uniformly.

[0217] 2.2 Experimental Results

[0218] (1) Appearance and visible foreign matters

[0219] When placed at 40°C ± 2°C for 10 days, the appearance of each group was clear to slightly opalescent, colorless to slightly yellow liquid, without foreign matters. That is, the appearance and visible foreign matters of each group were qualified.

[0220] (2) Protein content

[0221] The results of protein content detection are shown in Table 1. The results show that when placed at 40°C ± 2°C for 10 days, the protein content of each group of samples did not change significantly (change rate ≤ 10%).

[0222] Table 1. Results of protein content in pH screening (UV method, mg / ml)

[0223]

[0224]

[0225] (3) Purity

[0226] Purity (SEC-HPLC method): After being placed at 40±2℃ for 10 days, the purity of each group of samples did not change significantly (purity change value ≤1%). The results are shown in Table 2.

[0227] Purity (non-reducing CE-SDS method): After being placed at 40±2℃ for 10 days, the purity of each group of samples did not change significantly (purity change value ≤2%). The results are shown in Table 3.

[0228] Table 2. pH screening purity results (SEC-HPLC method, %)

[0229]

[0230] Table 3. pH screening purity results (non-reducing CE-SDS method, %)

[0231]

[0232] (4) Charge variants

[0233] The results of the charge variant (iCIEF method) are shown in Table 4, and their trends are shown in [the table below]. Figure 1 and Figure 2 The results showed that after being placed at 40℃±2℃ for 10 days, the changes in charge variants in each group of samples were mainly characterized by an increase in acidic components and a decrease in principal components. The changes in acidic components were 7.3%, 6.2%, 4.5%, and 7.7%, respectively, with smaller changes in acidic components in samples with pH 5.5-6.0. The changes in principal components were 7.8%, 6.0%, 4.8%, and 7.5%, respectively, with smaller changes in principal components in samples with pH 5.5-6.0.

[0234] Table 4. Results of pH screening for charge variants (iCIEF method, %)

[0235]

[0236] In summary, the pH screening results indicate that a pH between 5.5 and 6.0 is suitable for the citrate buffer system used in the antibody formulation. A pH of approximately 6.0 ± 0.3 was selected for the next round of formulation determination experiments.

[0237] Example 3. Prescription Determination Experiment

[0238] 3.1 Experimental Procedure

[0239] Based on the results of the pH screening experiment and the experience of the formulation development platform, the effects of different stabilizers (sorbitol, sucrose and arginine) on the stability of antibody proteins were investigated. A total of 4 formulations were designed, and detailed formulation information is shown in Table 5.

[0240] Table 5. Prescription Information Table

[0241] Serial Number Prescription information Prescription 1 1.55 mg / ml histidine, 80.00 mg / ml sucrose, 0.50 mg / ml polysorbate 80, pH 6.0 Prescription 2 1.55 mg / ml histidine, 50.00 mg / ml sorbitol, 0.50 mg / ml polysorbate 80, pH 6.0 Prescription 3 1.55 mg / ml histidine, 50.00 mg / ml sucrose, 0.50 mg / ml polysorbate 80, 13.94 mg / ml arginine, pH 6.0 Prescription 4 1.55 mg / ml histidine, 30.00 mg / ml sorbitol, 0.50 mg / ml polysorbate 80, 13.94 mg / ml arginine, pH 6.0

[0242] Prepare the buffer solutions for each formulation according to Table 5, adjust the pH to 6.0 with hydrochloric acid, and ultrafilter the antibody proteins into their respective formulation solutions. After replacement, adjust the protein concentration of each formulation to approximately 100 mg / ml, aseptically aliquot into 2R vials, cap, seal, and label, and then conduct accelerated experiments to obtain the optimal formulation. Experimental conditions and sampling plan are shown in Table 6.

[0243] Table 6. Experimental conditions and sampling plan

[0244] Serial Number Experimental conditions Experimental design and sampling points 1 40℃±2℃ Sampling was conducted on day 0, week 1, week 2, and month 1. 2 25℃±2℃ Sampling was conducted on day 0, in January, February, and March. 3 5℃±3℃ Sampling in the second month

[0245] 3.2 Experimental Results

[0246] (1) Appearance and visible foreign objects

[0247] Under three different temperature test conditions, the appearance and visible foreign matter of all four prescriptions were qualified.

[0248] (2) Protein content

[0249] Under the conditions of 40℃±2℃, 25℃±2℃ and 5℃±3℃, the protein content of the four prescriptions did not change significantly (change rate ≤10%), and the results are shown in Table 7.

[0250] Table 7. Protein content results of prescription screening experiment (UV method, mg / ml)

[0251]

[0252] (3) Purity

[0253] Purity (SEC-HPLC method): Under various temperature conditions, the purity of formulations 1 to 4 did not change significantly (purity change value ≤1%), and the results are shown in Table 8.

[0254] Purity (non-reducing CE-SDS method): Under various temperature conditions, the purity of formulations 1 to 3 did not change significantly (purity change value ≤2%). The concentration change of formulation 4 was greater than that of formulations 1 to 3 when accelerated at 25℃±2℃ for 3 months and when stored at 5℃±3℃ for 2 months. The results are shown in Table 9.

[0255] Table 8. Purity results of formulation screening experiments (SEC-HPLC method, %)

[0256]

[0257] Table 9. Purity results of formulation screening experiments (non-reducing CE-SDS method, %)

[0258]

[0259] (4) Charge variants

[0260] The results of charge variants (CEX-HPLC method) are shown in Table 10, and their trends are shown in [the table below]. Figure 3 and Figure 4 The results showed that under conditions of 40℃±2℃, the changes in charge variants in each group of samples mainly manifested as an increase in acidic components and a decrease in principal components. Specifically, the changes in acidic components (21%) and principal components (23.8%) in Formula 2 were less than those in Formula 1 (24.3%) and (26.1%), and the changes in acidic components (18.7%) and principal components (22.3%) in Formula 4 were less than those in Formula 3 (20.8%) and (23.6%). This indicates that sorbitol is superior to sucrose as a stabilizer for the antibody of this invention. After 3 months of accelerated storage at 25℃±2℃, changes in charge variants occurred, but there were no significant differences between Formula 1 and Formula 2, or between Formula 3 and Formula 4 (the difference in changes between formulas ≤2%). After 2 months of storage at 5℃±3℃, no significant changes occurred in the charge variants of any of the four formulas (the changes in principal components and acid / base components ≤2%).

[0261] Table 10. Results of charge variants in the formulation screening experiment (CEX-HPLC method, %)

[0262]

[0263]

[0264] Example 4. Process Validation Experiment

[0265] Based on the results of the experiments in Example 3, Formula 2 was selected as the optimal formula. Considering the requirements for stability and convenience in actual production processes, a fixed ratio of histidine hydrochloride and histidine was used to prepare the formula at pH 6.0±0.3. Specifically, the formula consists of 100 mg / ml recombinant anti-interleukin 23p19 subunit antibody, 0.76 mg / ml histidine, 1.08 mg / ml histidine hydrochloride, 50.00 mg / ml sorbitol, and 0.50 mg / ml polysorbate 80, at pH 6.0±0.3.

[0266] Three batches of the finished formulation were produced on a pilot-scale basis, with an actual pH value of 6.2 for all batches. Long-term stability was investigated at 5℃±3℃, with samples taken at 0, 3, 6, 9, and 12 months. The results are shown in Table 11. The results indicate that the protein in this formulation has good stability, with no significant differences between batches, and meets the quality standards of the actual production process.

[0267] Table 11. Results of Process Validation Experiments

[0268]

[0269] The foregoing describes exemplary embodiments of the present invention. Those skilled in the art should understand that these disclosures are merely exemplary, and various other substitutions, adaptations, and modifications can be made within the scope of the present invention. Therefore, the present invention is not limited to the specific embodiments listed herein. SEQUENCE LISTING <110> Innovent Biologics (Suzhou) Co., Ltd. <120> Preparations containing anti-IL-23p19 antibodies, their preparation methods and uses <130> P21404298C <150> CN2020104048344 <151> 2020-05-13 <160> 10 <170> PatentIn version 3.5 <210> 1 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> VH CDR1 <400> 1 Gly Tyr Thr Phe Thr Ser Tyr Leu Met His 1 5 10 <210> 2 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> VH CDR2 <400> 2 Tyr Ile Asn Pro Tyr Asn Glu Gly Thr Asn 1 5 10 <210> 3 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> VH CDR3 <400> 3 Asn Trp Asp Leu Pro Tyr 1 5 <210> 4 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> VL CDR1 <400> 4 Arg Ala Ser Gln Ser Ile Ser Asp Tyr Leu His 1 5 10 <210> 5 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> VL CDR2 <400> 5 Tyr Ala Ser Gln Ser Met Ser 1 5 <210> 6 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> VL CDR3 <400> 6 Gln Gln Gly His Ser Phe Pro Phe Thr 1 5 <210> 7 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 7 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Leu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Tyr Ile Asn Pro Tyr Asn Glu Gly Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asn Trp Asp Leu Pro Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 <210> 8 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> VL <400> 8 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Asp Tyr 20 25 30 Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Lys Tyr Ala Ser Gln Ser Met Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Ser Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly His Ser Phe Pro Phe 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 9 <211> 444 <212> PRT <213> Artificial Sequence <220> <223> HC <400> 9 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Leu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Tyr Ile Asn Pro Tyr Asn Glu Gly Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asn Trp Asp Leu Pro Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 115 120 125 Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val 130 135 140 Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala 145 150 155 160 Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly 165 170 175 Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly 180 185 190 Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys 195 200 205 Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys 210 215 220 Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu 225 230 235 240 Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr Ile Thr Arg Glu Pro Glu 245 250 255 Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys 260 265 270 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280 285 Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 290 295 300 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 305 310 315 320 Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys 325 330 335 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 340 345 350 Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 355 360 365 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 370 375 380 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 385 390 395 400 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 405 410 415 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 420 425 430 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 <210> 10 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> LC <400> 10 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Asp Tyr 20 25 30 Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Lys Tyr Ala Ser Gln Ser Met Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Ser Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly His Ser Phe Pro Phe 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210

Claims

1. A liquid antibody preparation comprising... (i) Anti-IL-23p19 antibody; (ii) Buffer, (iii) Stabilizers, and (iv) Surfactants, The anti-IL-23p19 antibody contains the following 6 CDRs: - Heavy chain VH CDR1 of GYTFTSYLMH (SEQ ID NO:1); - Heavy chain VH CDR2 of YINPYNEGTN (SEQ ID NO:2); - Heavy chain VH CDR3 of NWDLPY (SEQ ID NO:3); - RASQSISDYLH (SEQ ID NO:4) light chain VL CDR1; - The light chain VL CDR2 of YASQSMS (SEQ ID NO:5); and - The light chain VL CDR3 of QQGHSFPFT (SEQ ID NO:6), The CDR determines its boundaries using the AbM rule.

2. The liquid antibody preparation according to claim 1, characterized in that the pH of the liquid antibody preparation is 5.2-6.

3.

3. The liquid antibody preparation according to claim 1, characterized in that the pH of the liquid antibody preparation is 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2 or 6.

3.

4. The liquid antibody preparation according to claim 1, characterized in that the pH of the liquid antibody preparation is 6.0±0.

3.

5. The liquid antibody preparation according to any one of claims 1-4, characterized in that the concentration of anti-IL-23p19 antibody in the liquid antibody preparation is 25-250 mg / mL.

6. The liquid antibody preparation according to claim 5, characterized in that the concentration of anti-IL-23p19 antibody in the liquid antibody preparation is 50-200 mg / mL.

7. The liquid antibody formulation according to any one of claims 1-4 and 6, characterized in that the liquid antibody formulation comprises a histidine-histidine hydrochloride buffer system or a citrate-sodium citrate buffer system.

8. The liquid antibody formulation according to claim 7, wherein the buffer in the liquid antibody formulation is selected from histidine, histidine hydrochloride, and combinations thereof.

9. The liquid antibody formulation according to claim 7, characterized in that the buffer is selected from: (i) 0.775-3.1 mg / mL histidine or (ii) A combination of histidine and histidine hydrochloride, wherein the histidine content is 0.38-1.52 mg / mL and the histidine hydrochloride content is 0.54-2.16 mg / mL.

10. The liquid antibody formulation according to claim 7, characterized in that the buffer is selected from: (i) 1.55 mg / mL histidine, or (ii) A combination of histidine and histidine hydrochloride, wherein the concentrations of histidine and histidine hydrochloride are 0.76 mg / mL and 1.08 mg / mL, respectively.

11. The liquid antibody formulation according to any one of claims 1-4, 6 and 8-10, characterized in that the stabilizer is selected from: (i) 25-100 mg / ml of sorbitol; (ii) 40-160 mg / ml of sucrose; (iii) A combination containing sorbitol and arginine, wherein sorbitol is 15-60 mg / ml and arginine is 6.97-27.88 mg / ml; or (iv) A combination of sucrose and arginine, wherein the sucrose is 25-100 mg / ml and the arginine is 6.97-27.88 mg / ml.

12. The liquid antibody formulation according to claim 11, characterized in that the stabilizer is selected from: (i) 40-60 mg / ml of sorbitol; (ii) 70-90 mg / ml of sucrose; (iii) A combination comprising sorbitol and arginine, wherein sorbitol is 20-40 mg / ml and arginine is 10.45-17.42 mg / ml; or (iv) A combination of sucrose and arginine, wherein sucrose is 40-60 mg / ml and arginine is 10.45-17.42 mg / ml.

13. The liquid antibody formulation according to any one of claims 1-4, 6, 8-10 and 12, characterized in that the surfactant in the liquid antibody formulation is selected from polysorbate surfactants, poloxamer, polyethylene glycol or combinations thereof.

14. The liquid antibody formulation according to claim 13, characterized in that the surfactant in the liquid antibody formulation is polysorbate-80 or polysorbate-20.

15. The liquid antibody formulation according to claim 11, characterized in that the surfactant in the liquid antibody formulation is selected from polysorbate surfactants, poloxamer, polyethylene glycol, or combinations thereof.

16. The liquid antibody preparation according to any one of claims 1-4, 6, 8-10, 12, 14 and 15, characterized in that the concentration of the surfactant is 0.1-1 mg / ml.

17. The liquid antibody formulation according to claim 16, characterized in that the concentration of the surfactant is 0.2-0.8 mg / ml.

18. The liquid antibody formulation according to claim 16, characterized in that the concentration of the surfactant is 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 or 0.8 mg / ml.

19. The liquid antibody formulation according to any one of claims 1-4, 6, 8-10, 12, 14, 15, 17 and 18, characterized in that the anti-IL-23p19 antibody comprises a heavy chain variable region VH and a light chain variable region VL, wherein the heavy chain variable region comprises the sequence of SEQ ID NO: 7 or a sequence having at least 90% identity with it, and the light chain variable region comprises the sequence of SEQ ID NO: 8 or a sequence having at least 90% identity with it.

20. The liquid antibody formulation according to claim 19, characterized in that the anti-IL-23p19 antibody comprises a heavy chain variable region VH and a light chain variable region VL, wherein the heavy chain variable region comprises the sequence of SEQ ID NO: 7 or a sequence having at least 99% identity with it, and the light chain variable region comprises the sequence of SEQ ID NO: 8 or a sequence having at least 99% identity with it.

21. The liquid antibody formulation according to claim 20, characterized in that the anti-IL-23p19 antibody comprises a heavy chain variable region VH and a light chain variable region VL, wherein the heavy chain variable region comprises the sequence of SEQ ID NO: 7, and the light chain variable region comprises the sequence of SEQ ID NO:

8.

22. The liquid antibody preparation according to any one of claims 1-4, 6, 8-10, 12, 14, 15, 17, 18, 20 and 21, characterized in that the anti-IL-23p19 antibody is an IgG1 type antibody.

23. The liquid antibody formulation according to claim 22, characterized in that the anti-IL-23p19 antibody comprises a heavy chain sequence having at least 90% identity with SEQ ID NO:9 and a light chain sequence having at least 90% identity with SEQ ID NO:

10.

24. The liquid antibody formulation according to claim 23, characterized in that the anti-IL-23p19 antibody comprises a heavy chain sequence of SEQ ID NO:9 or having at least 99% identity with it and a light chain sequence of SEQ ID NO:10 or having at least 99% identity with it.

25. The liquid antibody formulation according to claim 23, characterized in that the anti-IL-23p19 antibody comprises the heavy chain sequence of SEQ ID NO:9 and the light chain sequence of SEQ ID NO:

10.

26. The liquid antibody formulation according to any one of claims 1-4, 6, 8-10, 12, 14, 15, 17, 18, 20, 21, 24 and 25, characterized in that the anti-IL-23p19 antibody is recombinantly expressed in HEK 293 cells or CHO cells.

27. The liquid antibody preparation according to any one of claims 1-4, 6, 8-10, 12, 14, 15, 17, 18, 20, 21, 24 and 25, characterized in that the liquid antibody preparation is an injection or an infusion.

28. The liquid antibody preparation according to claim 27, characterized in that the injectable preparation is for subcutaneous or intravenous injection.

29. The liquid antibody preparation according to claim 27, characterized in that the infusion solution is for intravenous infusion.

30. The liquid antibody formulation according to any one of claims 1-4, 6, 8-10, 12, 14, 15, 17, 18, 20, 21, 24, 25, 28 and 29, comprising: (i) Anti-IL-23p19 antibody at a concentration of 50-200 mg / ml; (ii) Histidine at concentrations of 0.775–3.1 mg / mL; (iii) 40-60 mg / mL of sorbitol; and (iv) 0.2-0.8 mg / ml of polysorbate 80; The pH of the liquid antibody preparation is 6.0 ± 0.3; Alternatively, the liquid antibody formulation contains (i) Anti-IL-23p19 antibody at a concentration of 50-200 mg / ml; (ii) Histidine at concentrations of 0.775–3.1 mg / mL; (iii) 20-40 mg / mL of sorbitol, 10.45-17.42 mg / mL of arginine; and (iv) 0.2-0.8 mg / ml of polysorbate 80; The pH of the liquid antibody preparation is 6.0 ± 0.3; Alternatively, the liquid antibody formulation contains (i) Anti-IL-23p19 antibody at a concentration of 50-200 mg / ml; (ii) Histidine buffer at a concentration of 0.775–3.1 mg / mL; (iii) 70-90 mg / ml of sucrose; and (iv) 0.2-0.8 mg / ml of polysorbate 80; The pH of the liquid antibody preparation is 6.0 ± 0.3; Alternatively, the liquid antibody formulation contains (i) Anti-IL-23p19 antibody at a concentration of 50-200 mg / ml; (ii) Histidine at concentrations of 0.775–3.1 mg / mL; (iii) 40-60 mg / ml sucrose, 10.45-17.42 mg / ml arginine; and (iv) 0.2-0.8 mg / ml of polysorbate 80; The pH of the liquid antibody preparation is 6.0 ± 0.3; Alternatively, the liquid antibody formulation contains (i) Anti-IL-23p19 antibody at a concentration of 50-200 mg / ml; (ii) 0.38-1.52 mg / mL histidine, 0.54-2.16 mg / mL histidine hydrochloride; (iii) 40-60 mg / mL of sorbitol; and (iv) 0.2-0.8 mg / ml of polysorbate 80; The pH of the liquid antibody preparation is 6.0 ± 0.3; Alternatively, the liquid antibody formulation contains (i) Anti-IL-23p19 antibody at a concentration of 50-200 mg / ml; (ii) 0.38-1.52 mg / mL histidine, 0.54-2.16 mg / mL histidine hydrochloride; (iii) 20-40 mg / mL of sorbitol, 10.45-17.42 mg / mL of arginine; and (iv) 0.2-0.8 mg / ml of polysorbate 80; The pH of the liquid antibody preparation is 6.0 ± 0.3; Alternatively, the liquid antibody formulation contains (i) Anti-IL-23p19 antibody at a concentration of 50-200 mg / ml; (ii) 0.38-1.52 mg / mL histidine, 0.54-2.16 mg / mL histidine hydrochloride; (iii) 70-90 mg / ml of sucrose; and (iv) 0.2-0.8 mg / ml of polysorbate 80; The pH of the liquid antibody preparation is 6.0 ± 0.3; Alternatively, the liquid antibody formulation contains (i) Anti-IL-23p19 antibody at a concentration of 50-200 mg / ml; (ii) 0.38-1.52 mg / mL histidine, 0.54-2.16 mg / mL histidine hydrochloride; (iii) 40-60 mg / ml sucrose, 10.45-17.42 mg / ml arginine; and (iv) 0.2-0.8 mg / ml of polysorbate 80; The pH of the liquid antibody preparation is 6.0 ± 0.

3.

31. The liquid antibody formulation of claim 30, comprising 50, 100, 150 or 200 mg / ml of anti-IL-23p19 antibody.

32. The liquid antibody formulation according to claim 30, wherein the pH of the liquid antibody formulation is 6.

0.

33. The liquid antibody formulation according to claim 30, comprising: (i) Anti-IL-23p19 antibody at a concentration of 50-200 mg / ml; (ii) Histidine at concentrations of 0.775–3.1 mg / mL; (iii) 40-60 mg / mL of sorbitol; and (iv) 0.2-0.8 mg / ml of polysorbate 80; The pH of the liquid antibody preparation is 6.0 ± 0.

3.

34. The liquid antibody formulation according to claim 30, comprising: (i) Anti-IL-23p19 antibody at a concentration of 50-200 mg / ml; (ii) 0.38-1.52 mg / mL histidine, 0.54-2.16 mg / mL histidine hydrochloride; (iii) 40-60 mg / mL of sorbitol; and (iv) 0.2-0.8 mg / ml of polysorbate 80; The pH of the liquid antibody preparation is 6.0 ± 0.

3.

35. A solid antibody formulation obtained by solidifying the liquid antibody formulation according to any one of claims 1-34.

36. The solid antibody formulation according to claim 35, wherein the solid antibody formulation is in the form of a lyophilized powder for injection.

37. A delivery device comprising a liquid antibody formulation of any one of claims 1-34 or a solid antibody formulation of claim 35 or 36.

38. A pre-filled syringe comprising a liquid antibody formulation of any one of claims 1-34 or a solid antibody formulation of claim 35 or 36, for intravenous or intramuscular injection.

39. Use of the liquid antibody formulation according to any one of claims 1-34 or the solid antibody formulation according to claim 35 or 36 for the preparation of a medicament for treating immune system diseases or inflammation.

40. The use according to claim 39, wherein the immune system disease is an autoimmune disease.

41. Use of the liquid antibody formulation of any one of claims 1-34 or the solid antibody formulation of claim 35 or 36 for the preparation of a delivery device for treating immune system diseases or inflammation.

42. The use according to claim 41, wherein the immune system disease is an autoimmune disease.

43. Use of the liquid antibody formulation of any one of claims 1-34 or the solid antibody formulation of claim 35 or 36 for the preparation of a prefilled syringe for treating immune system diseases or inflammation.

44. The use according to claim 43, wherein the immune system disease is an autoimmune disease.

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