Il-12-albumin binding domain fusion protein formulations and methods of use thereof

Abstract

The present disclosure provides a formulation of an IL-12-albumin binding domain (ABD) fusion protein comprising from about 10 [mu] g / mL to about 50 [mu] g / mL fusion protein, from about 25 mM to about 100 mM alanine, from about 100 mM to about 400 mM trehalose, from about 10 mM to about 50 mM glycylglycine, from about 0.01% to about 0.04% polysorbate 20 (v / v), and from about 5 [mu] M to about 20 [mu] M diethylenetriamine pentaacetic acid (DTPA) wherein the pH of the formulation is from about 6.8 to about 8.0. Such formulations may be used to treat and / or prevent IL-12 related diseases or conditions in a subject in need thereof.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 346,368, filed on May 27, 2023, which is incorporated herein by reference in its entirety. Technical Field

[0003] The present disclosure generally provides IL-12-albumin-binding domain (ABD) fusion protein preparations and methods of using the same, including for treating IL-12-related diseases and / or disorders. Background Art

[0004] IL-12 can mediate immune effector functions in a manner consistent with enhancing proinflammatory, endogenous anti-tumor immune responses. (See, e.g., Boggio et al., J Exp Med 188:589-96 (1998); Cavallo et al., Cancer Res 59:414-21 (1999); Yu et al. Int Immuno L8:855-65 (1996); Nastala et al., J Immunol 153:1697:706 (1994); Brunda et al., J Exp Med 178:1223-30 (1993)). IL-12 is known to induce inflammatory Th1 CD4+ T cell responses and enhance the cytotoxicity of CD8+ T cells. Studies have also shown that IFNγ secretion by T cells mediated by IL-12 can reverse T cell anergy and cause effector T cells to resist immunosuppressive regulatory T cells. IL-12 can not only activate the adaptive and innate immune systems, but also further modulate the originally immune-hostile tumor microenvironment, making it an ideal candidate for tumor immunotherapy.

[0005] Serum albumin has a long half-life of 2 to 4 weeks due to recycling through the neonatal Fc receptor (FcRn). Albumin is taken up by endothelial cells by macropinocytosis and binds to FcRn in a pH-dependent manner in the acidic environment of early endosomes. Albumin-FcRn binding avoids degradation of albumin molecules in lysosomal compartments and redirects albumin molecules to the plasma membrane where they are released back into the plasma due to the neutral pH.

[0006] The albumin binding domain (ABD) fusion protein described in U.S. Patent No. 11,028,166 can be used to extend the half-life of biological agents (such as interleukins and antibodies). Such ABD will not compete with FcRN for albumin binding, and binds to albumin within a specific pH range, which allows ABD to also carry out FcRn-driven endosomal albumin recycling when bound to albumin. Therefore, biological agents comprising ABD can use the albumin-FcRn pathway to escape lysosomal degradation, and therefore, compared with biological agents lacking ABD, their serum half-life is longer. In addition, such therapeutic agents containing ABD are advantageously positioned at tumors known to contain high levels of serum albumin. Therefore, such therapeutic agents containing ABD are particularly suitable for treating cancer.

[0007] Formulations that maintain the stability of the fusion protein under various conditions are beneficial for the therapeutic use of ABD fusion proteins. Importantly, therapeutic formulations allow storage without unacceptable loss of activity of the active protein, minimize the accumulation of undesirable products such as aggregates or degradation materials (e.g., fragments, oxidation, deamidation or isomerization materials), provide appropriate protein concentrations, and do not contain components that are incompatible with therapeutic applications. Therefore, there is a need in the art for a stable pharmaceutical formulation comprising an IL-12-ABD fusion protein that is suitable for therapeutic use. Summary of the invention

[0008] The present disclosure provides an IL12-ABD fusion protein formulation that has unique stability in both liquid and solid (e.g., lyophilized) forms when the concentration of the fusion protein is low. This stable liquid pharmaceutical formulation comprises about 10 μg / mL to about 50 μg / mL of an IL12-ABD fusion protein of SEQ ID NO: 11, about 25 mM to about 100 mM alanine, about 100 mM to about 400 mM trehalose, about 10 mM to about 50 mM glycylglycine, about 0.01% to about 0.04% polysorbate 20 (v / v); and about 5 μM to about 20 μM diethylenetriaminepentaacetic acid (DTPA), wherein the pH of the formulation is about 6.8 to about 8.0.

[0009] In some embodiments, the liquid formulation comprises a fusion protein of SEQ ID NO: 11 in an amount of about 10 μg / mL to about 30 μg / mL, about 15 μg / mL to about 25 μg / mL, about 10 μg / mL to about 15 μg / mL, about 15 μg / mL to about 20 μg / mL, about 20 μg / mL to about 25 μg / mL, about 25 μg / mL to about 30 μg / mL, about 30 μg / mL to about 35 μg / mL, about 35 μg / mL to about 40 μg / mL, about 40 μg / mL to about 45 μg / mL, about 45 μg / mL to about 50 μg / mL, about 10 μg / mL, about 15 μg / mL, about 20 μg / mL, about 25 μg / mL, about 30 μg / mL, about 35 μg / mL, about 40 μg / mL, about 45 μg / mL and / or about 50 μg / mL.

[0010] In some embodiments, the liquid formulation comprises the fusion protein of SEQ ID NO: 11 in an amount of about 10 μg / mL to about 30 μg / mL.

[0011] In some embodiments, the liquid formulation comprises about 20 μg / mL of the fusion protein of SEQ ID NO: 11.

[0012] In some embodiments, the liquid formulation comprises about 25 mM to about 75 mM alanine, about 40 mM to about 60 mM alanine, about 25 mM to about 30 mM alanine, about 30 mM to about 40 mM alanine, about 40 mM to about 50 mM alanine, about 50 mM to about 60 mM alanine, about 60 mM to about 70 mM alanine, about 70 mM to about 80 mM alanine, about 80 mM to about 90 mM alanine, about 90 mM to about 100 mM alanine, about 25 mM alanine, about 30 mM alanine, about 40 mM alanine, about 50 mM alanine, about 60 mM alanine, about 70 mM alanine, about 80 mM alanine, about 90 mM alanine and / or about 100 mM alanine.

[0013] In some embodiments, the liquid formulation comprises about 50 mM alanine.

[0014] In some embodiments, the liquid formulation comprises about 100 mM to about 300 mM trehalose, about 150 mM to about 250 mM trehalose, about 100 mM to about 150 mM trehalose, about 150 mM to about 200 mM trehalose, about 200 mM to about 250 mM trehalose, about 250 mM to about 300 mM trehalose, about 300 mM to about 350 mM trehalose, about 350 mM to about 400 mM trehalose, about 100 mM trehalose, about 150 mM trehalose, about 200 mM trehalose, about 250 mM trehalose, about 300 mM trehalose, about 350 mM to about 400 mM trehalose.

[0015] In some embodiments, the liquid formulation comprises about 200 mM trehalose.

[0016] In some embodiments, the liquid formulation comprises about 10 mM to about 40 mM glycylglycine, about 10 mM to about 30 mM glycylglycine, about 10 mM to about 15 mM glycylglycine, about 15 mM to about 20 mM glycylglycine, about 20 mM to about 25 mM glycylglycine, about 25 mM to about 30 mM glycylglycine, about 35 mM to about 40 mM glycylglycine, about 4 0 mM to about 45 mM glycylglycine, about 45 mM to about 50 mM glycylglycine, about 10 mM glycylglycine, about 15 mM glycylglycine, about 20 mM glycylglycine, about 25 mM glycylglycine, about 30 mM glycylglycine, about 35 mM glycylglycine, about 40 mM glycylglycine, about 45 mM glycylglycine and / or about 50 mM glycylglycine.

[0017] In some embodiments, the liquid formulation comprises about 25 mM glycylglycine.

[0018] In some embodiments, the liquid formulation comprises about 0.01% to about 0.03% polysorbate 20 (v / v), about 0.01% to about 0.02% polysorbate 20 (v / v), about 0.02% to about 0.03% polysorbate 20 (v / v), about 0.03% to about 0.04% polysorbate 20 (v / v), about 0.01% polysorbate 20 (v / v), about 0.02% polysorbate 20 (v / v), about 0.03% polysorbate 20 (v / v), and / or about 0.04% polysorbate 20 (v / v).

[0019] In some embodiments, the liquid formulation comprises about 0.02% polysorbate 20 (v / v).

[0020] In some embodiments, the liquid formulation comprises about 5 μM to about 15 μM DTPA, about 5 μM to about 10 μM DTPA, about 10 μM to about 15 μM DTPA, about 15 μM to about 20 μM DTPA, about 5 μM DTPA, about 10 μM DTPA, about 15 μM DTPA, and / or about 20 μM DTPA.

[0021] In some embodiments, the liquid formulation comprises about 10 μM DTPA.

[0022] In some embodiments, the liquid formulation has a pH of about 7.0 to about 7.8, about 7.2 to about 7.8, about 7.4 to about 7.6, about 7.0 to about 7.2, about 7.2 to about 7.4, about 7.4 to about 7.6, about 7.6 to about 7.8, about 7.8 to about 8.0, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, and / or about 8.0.

[0023] In some embodiments, the liquid formulation has a pH of about 7.5.

[0024] In some embodiments, the liquid drug formulation comprises about 20 μg / mL of a fusion protein of SEQ ID NO: 11, about 50 mM alanine, about 200 mM trehalose, about 25 mM glycylglycine, about 0.02% polysorbate 20 (v / v), and about 10 μM DTPA, wherein the formulation has a pH of about 7.5.

[0025] In some embodiments, the liquid formulation is a reconstituted formulation.

[0026] In some embodiments, the reconstituted formulation is reconstituted from a lyophilized formulation.

[0027] The stable solid pharmaceutical formulation comprises about 0.01% to about 0.03% (w / w) of the IL12-ABD fusion protein of SEQ ID NO: 11, about 2% to about 8% alanine (w / w), about 50% to about 95% trehalose (w / w), about 2% to about 8% glycylglycine (w / w), about 0.1% to about 0.4% polysorbate 20 (w / w), and about 0.002% to about 0.006% diethylenetriaminepentaacetic acid (DTPA) (w / w).

[0028] In some embodiments, the solid pharmaceutical formulation comprises about 0.024 to about 0.028% (w / w) of the fusion protein of SEQ ID NO: 11, about 4.0% to about 5.0% alanine (w / w), about 70% to about 80% trehalose (w / w), about 3.0% to about 4.0% glycylglycine (w / w), about 0.15% to about 0.30% polysorbate 20 (w / w), and about 0.0045% to about 0.0055% DTPA (w / w).

[0029] In some embodiments, the solid formulation is a lyophilized formulation.

[0030] In some embodiments, the formulation is suitable for parenteral administration.

[0031] In some embodiments, the formulations are suitable for intravenous administration.

[0032] In some embodiments, the formulation is suitable for subcutaneous administration.

[0033] The present disclosure also provides a method of treating an IL-12-related disease or disorder in a subject in need thereof, the method comprising administering to the subject a formulation as disclosed herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The sequence of the albumin binding domain contained in the subject albumin binding domain fusion protein described herein is shown. The figure includes the variable heavy chain domain sequence and the variable light chain domain sequence, as well as the vhCDR1-3 sequence and the vlCDR1-3 sequence.

[0035] Figure 2 Provided is the amino acid sequence of the IL-12 cytokine contained in the ABD fusion proteins described herein. Figure 2 Also described are IL12-ABD fusion proteins, wherein the ABD is A10m3. DETAILED DESCRIPTION

[0036] 1.1 Definition

[0037] The following definitions are included to provide a clear and consistent understanding of the specification and claims. As used herein, the referenced terms have the following meanings. All other terms and phrases used in this specification have the common meanings understood by those skilled in the art. These common meanings can be obtained by reference to technical dictionaries, such as Hawley's Condensed Chemical Dictionary 14th Edition, by RJ Lewis, John Wiley & Sons, New York, NY, 2001.

[0038] References in the specification to "one embodiment", "embodiment", etc. mean that the embodiment may include specific aspects, features, structures, parts or characteristics, but not every embodiment necessarily includes such aspects, features, structures, parts or characteristics. In addition, such phrases may (but do not necessarily) refer to the same embodiment mentioned in other parts of the specification. In addition, when specific aspects, features, structures, parts or characteristics are described in conjunction with an embodiment, those skilled in the art will be aware of the effects or connections between such aspects, features, structures, parts or characteristics and other embodiments (whether or not explicitly described).

[0039] The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a formulation" includes a plurality of such formulations, and thus formulation X includes a plurality of formulations X. It is also noted that the claims may be drafted to exclude any optional elements. Therefore, this statement is intended to serve as antecedent basis for use of exclusive terminology (e.g., "only," "only," etc.) in connection with any elements described herein, and / or recitation or use of "negative" limitations with respect to claim elements.

[0040] The term "and / or" means any one item, any combination of items, or all items associated with the term. The phrases "one or more" and "at least one" are easily understood by those skilled in the art, especially when reading the context in which they are used. For example, the phrase can mean one, two, three, four, five, six, ten, 100, or any upper limit.

[0041] A "stable" IL12-ABD fusion protein formulation refers to a formulation in which the IL12-ABD fusion protein substantially maintains its physical stability and / or chemical stability and / or its biological activity during storage. The storage period is typically selected based on the expected shelf life of the formulation. There are a variety of analytical techniques in the art for measuring protein stability. Examples of analytical techniques are described below. "Substantially maintained" refers to a retention rate of 85% or more, such as a retention rate of at least 90% or a retention rate of at least 95%.

[0042] An IL12-ABD fusion protein "maintains its physical stability" in a pharmaceutical formulation if the IL12-ABD fusion protein does not exhibit significant physical changes (e.g., aggregation, precipitation, and / or denaturation) when visually inspected for color and / or clarity, or when measured by ultraviolet light scattering or size exclusion chromatography.

[0043] If the IL12-ABD fusion protein does not show significant chemical changes, the IL12-ABD fusion protein "maintains its chemical stability" in a pharmaceutical formulation. Chemical stability can be assessed by detecting and quantifying chemically altered forms of the protein. Chemical changes can involve size changes (e.g., shearing), which can be evaluated, for example, using size exclusion chromatography, SDS-PAGE, and / or matrix-assisted laser desorption ionization / time-of-flight mass spectrometry (MALDI / TOF MS). Other types of chemical changes include charge changes (e.g., due to deamidation, oxidation, and / or isomerization), which can be evaluated, for example, by ion exchange chromatography.

[0044] If the biological activity of the IL12-ABD fusion protein does not change significantly over a given period of time compared to the biological activity exhibited when the pharmaceutical formulation was prepared, the IL12-ABD fusion protein "retains its biological activity" in a pharmaceutical formulation. The "biological activity" of an IL12-ABD fusion protein refers to the ability of the protein to produce a measurable biological response, which can be measured in vitro or in vivo.

[0045] A "pharmaceutical formulation" refers to a preparation that does not contain ingredients in amounts that are unacceptably toxic to a patient to whom the preparation would be administered.

[0046] The percentage "identity" between a polypeptide sequence and a reference sequence is defined as the percentage of amino acid residues in the polypeptide sequence that are identical to the amino acid residues in the reference sequence, after the sequences are aligned and gaps are introduced (if necessary) to achieve maximum percentage sequence identity. Alignments for determining percentage amino acid sequence identity can be achieved in various ways well known to those skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, CLUSTAL OMEGA or MUSCLE software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm required for achieving maximum alignment over the entire length of the compared sequences. Unless otherwise indicated, the BLAST algorithm using default parameters determines percentage sequence identity.

[0047] A "subject" refers to a human or non-human mammal, including but not limited to bovine, equine, canine, ovine, feline, and rodent (including murine and house mouse species) subjects. A "patient" is a human subject.

[0048] As used herein, the terms "treat", "treating", "treatment" and the like refer to the alleviation or improvement of a disease and / or the signs or symptoms associated therewith, or the slowing down or arresting of its progression. It should be understood that, although not excluded, the treatment of a disease or disorder does not require the complete elimination of the disease, disorder or symptoms associated therewith.

[0049] In this disclosure, the words "comprises," "comprising," "containing," "having," "includes," "including," and variations thereof have the meanings ascribed to them in U.S. patent law, allowing for the presence of additional components other than those explicitly recited.

[0050] Those skilled in the art will appreciate that all numbers, including those representing the amount of ingredients, properties (e.g., molecular weight), reaction conditions, etc., are approximate values ​​and are understood to be optionally modified with the term "about" in all cases. These values ​​may vary, depending on the desired properties sought to be obtained by those skilled in the art using the teachings described herein. It is also understood that these values ​​inherently contain variability caused by the standard deviations that must occur in their respective test measurements. When the preposition "about" is used to represent a value as an approximation, it is understood that the specific value without the modifier "about" also forms another aspect.

[0051] The terms "about" and "approximately" are used interchangeably. Both terms can refer to a variation of ±1%, ±5%, or ±10% of a specified value. For example, "about 50%" can have a variation from 45% to 55% in some embodiments, or as defined in a particular claim. For integer ranges, the term "about" can include one or two integers that are greater than and / or less than the integers at both ends of the range. Unless otherwise specified herein, the terms "about" and "approximately" are intended to include values ​​close to the range, such as weight percentages, which are equivalent in terms of the functionality of a single component, composition, or embodiment. The terms "about" and "approximately" can also modify the endpoints of the ranges described above in this paragraph.

[0052] Those skilled in the art will understand that for any and all purposes, especially in providing written descriptions, all ranges described herein also encompass any and all possible sub-ranges and combinations of their sub-ranges, as well as single values, particularly integer values, that make up the ranges. Therefore, it should be understood that each unit between two specific units is also disclosed. For example, if 10 to 15 is disclosed, 11, 12, 13, and 14 are also disclosed separately and as part of the range. The range (e.g., weight percentage or carbon group) described includes each specific value, integer, decimal, or identity within the range. It can be easily recognized that any listed range is sufficient to describe and allow the same range to be decomposed into at least equal halves, thirds, quarters, fifths, or tenths. As a non-limiting example, each range discussed herein can be easily decomposed into the lower third, the middle third, and the upper third, etc. It will also be understood by those skilled in the art that all languages, such as "at most", "at least", "greater than", "less than", "more than", "or more", etc., include the numbers listed, and these terms refer to ranges that can be subsequently decomposed into sub-ranges as described above. In the same manner, all ratios listed herein also include all sub-ratios that fall within a wider ratio. Therefore, the specific values ​​recited for radicals, substituents, and ranges are for illustration only; they do not exclude other defined values ​​or other values ​​within the defined ranges of radicals, substituents, and ranges. It will be further understood that the endpoints of each range are significant relative to the other endpoint, and independent of the other endpoint.

[0053] The present disclosure provides ranges, limits and deviations of variables (e.g., volume, mass, percentage, ratio, etc.). It will be understood by those of ordinary skill in the art that ranges (e.g., "number 1" to "number 2") are meant to include a continuous range of integers and fractions. For example, 1 to 10 means 1, 2, 3, 4, 5, ..., 9, 10. It also means 1.0, 1.1, 1.2, 1.3, ..., 9.8, 9.9, 10.0, and also means 1.01, 1.02, 1.03, etc. If the disclosed variable is a number less than "number 10", it means a continuous range of integers and fractions less than number 10, as described above.

[0054] Similarly, if a variable is disclosed as a number greater than "the number 10," it means a continuous range that includes integers and fractions greater than the number 10.

[0055] These ranges may be modified by the term "about", the meaning of which has been described above.

[0056] It is also readily recognized by those skilled in the art that when members are grouped together in a common manner, such as in a Markush group, the present invention encompasses not only the entire group listed as a whole, but also each individual member of the group and all possible subgroups of the main group. Furthermore, for all purposes, the present invention encompasses not only the main group, but also the main group lacking one or more group members. Thus, the present invention contemplates the explicit exclusion of any one or more members of the group. Thus, any disclosed class or embodiment may be subject to an appendix in which any one or more of the elements, species or embodiments may be excluded from such class or embodiment, for example, for explicit negative limitation.

[0057] IL12-ABD fusion protein

[0058] Interleukin 12 (also known as IL-12 or IL12) refers to a heterodimeric cytokine interleukin encoded by the IL-12A and IL-12B genes (Genbank accession numbers: NM_000882 (IL-12A) and NM_002187 (IL-12B)). IL-12 consists of a bundle of four alpha helices and is involved in the differentiation of natural T cells into TH1 cells. IL-12 binds to the IL-12 receptor, which is a heterodimeric receptor formed by IL-12R-β1 and IL-12R-β2. IL-12 is known as a T cell stimulatory factor that can stimulate the growth and function of T cells. In particular, IL-12 can stimulate T cells and natural killer (NK) cells to produce interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α), and reduce IL-4-mediated IFN-γ inhibition. IL-12 can also mediate an enhancement of the cytotoxic activity of NK cells and CD8+ cytotoxic T lymphocytes. In addition, IL-12 can also have anti-angiogenic activity by increasing the production of interferon gamma, which in turn increases the production of chemokine-induced protein 10 (IP-10 or CXCL10). IP-10 then mediates this anti-angiogenic effect. Without being bound by any particular theory of operation, it is believed that IL-12 can be used to treat cancer through its ability to induce an immune response and its anti-angiogenic activity.

[0059] In an embodiment, IL-12 is a single-chain IL-12 polypeptide comprising an IL-12p35 subunit linked to an IL-12p40 subunit. Such IL-12 single-chain polypeptides advantageously retain one or more biological activities of wild-type IL-12. In some embodiments, the single-chain IL-12 polypeptides described herein have the following chemical formula from N-terminus to C-terminus: (p40)-(L)-(p35), wherein "p40" is an IL-12p40 subunit, "p35" is an IL-12p35 subunit, and L is a linker. In other embodiments, the single-chain IL-12 has the following chemical formula from N-terminus to C-terminus: (p35)-(L)-(p40). Any suitable linker can be used for a single-chain IL-12 polypeptide, including those described herein. Suitable linkers can include, for example, a linker having the amino acid sequence (GGGGS) x , wherein x is an integer from 1 to 10. Other suitable linkers include, for example, the amino acid sequence GGGGGGS. Exemplary single-chain IL-12 linkers that can be used for the subject single-chain IL-12 polypeptides are also described in Lieschke et al., Nature Biotechnology 15:35-40 (1997), which is incorporated herein by reference in its entirety, particularly for its teachings on IL-12 polypeptide linkers.

[0060] The IL12-ABD fusion proteins provided herein comprise an albumin binding domain connected to an IL12 fusion partner via a linker. As described herein, the subject ABD fusion proteins are capable of FcRn-mediated endosomal recycling and therefore advantageously exhibit an extended half-life compared to counterparts that do not comprise such an ABD. For example, in Figure 1 The amino acid sequence of ABD, including vhCDR1, vhCDR2, vhCDR3, vlCDR1, vlCDR2, vlCDR3, variable heavy chain sequence and variable light chain sequence, is disclosed in.

[0061] 1.3. Preparation

[0062] In one aspect, an aqueous liquid or solid preparation (pharmaceutical composition) is provided herein, such as a lyophilized preparation, comprising an IL12-ABD fusion protein. In one embodiment, the preparation is a reconstituted preparation reconstituted by a lyophilized preparation. In one embodiment, the preparation is sterile. In one embodiment, the preparation is stable. In one embodiment, the preparation further comprises at least one free amino acid. In one embodiment, the preparation further comprises at least one surfactant. In one embodiment, the preparation further comprises at least one buffer component. In one embodiment, the preparation further comprises at least one chelating agent. In one embodiment, the preparation comprises at least one sugar. In one embodiment, the preparation comprises an IL12-ABD fusion protein. In one embodiment, the preparation comprises at least one free amino acid, at least one surfactant, at least one buffer component, at least one chelating agent, at least one sugar and an IL12-ABD fusion protein.

[0063] In one embodiment, the liquid formulation comprises about 10 μg / mL to about 50 μg / mL of IL12-ABD fusion protein, about 25 mM to about 100 mM alanine, about 100 mM to about 400 mM trehalose, about 10 mM to about 50 mM glycylglycine, about 0.01% to about 0.04% polysorbate 20 (v / v), and about 5 μM to about 20 μM diethylenetriaminepentaacetic acid (DTPA), wherein the pH of the formulation is about 6.8 to about 8.0. In one embodiment, the liquid formulation comprises about 20 μg / mL to about 50 μg / mL of IL12-ABD fusion protein, about 50 mM alanine, about 200 mM trehalose, about 25 mM glycylglycine, about 0.02% polysorbate 20 (v / v), and about 10 μM DTPA, wherein the pH of the formulation is about 7.5. In one embodiment, the liquid formulation comprises about 20 μg / mL of IL12-ABD fusion protein, about 50 mM alanine, about 200 mM trehalose, about 25 mM glycylglycine, about 0.02% polysorbate 20 (v / v), and about 10 μM DTPA, wherein the pH of the formulation is about 7.5.

[0064] In one embodiment, the solid formulation comprises about 0.01% to about 0.03% (w / w) of the fusion protein, about 2% to about 8% alanine (w / w), about 50% to about 95% trehalose (w / w), about 2% to about 8% glycylglycine (w / w), about 0.1% to about 0.4% polysorbate 20 (w / w), and about 0.002% to about 0.006% diethylenetriaminepentaacetic acid (DTPA) (w / w). In one embodiment, the solid formulation comprises about 0.024 to about 0.028% (w / w) of the fusion protein, about 4.0% and about 5.0% alanine (w / w), about 70% to about 80% trehalose (w / w), about 3.0% to about 4.0% glycylglycine (w / w), about 0.15% to about 0.30% polysorbate 20 (w / w), and about 0.0045% to about 0.0055% DTPA (w / w). In one embodiment, the weight percentages are w / w of the lyophilized formulation.

[0065] 1.3.1. IL12-ABD fusion protein concentration

[0066] In one embodiment, the concentration of IL12-ABD in the liquid formulation is about 10 μg / mL to about 50 μg / mL, about 10 μg / mL to about 30 μg / mL, about 15 μg / mL to about 25 μg / mL, about 10 μg / mL to about 15 μg / mL, about 15 μg / mL to about 20 μg / mL, about 20 μg / mL to about 25 μg / mL, about 25 μg / mL to about 30 μg / mL, about 30 μg / mL to about 35 μg / mL, about 35 μg / mL to about 40 μg / mL, about 40 μg / mL to about 45 μg / mL, about 45 μg / mL to about 50 μg / mL, about 10 μg / mL, about 15 μg / mL, about 20 μg / mL, about 25 μg / mL, about 30 μg / mL, about 35 μg / mL, about 40 μg / mL, about 45 μg / mL and / or about 50 μg / mL.

[0067] In one embodiment, the concentration of IL12-ABD in the liquid formulation is about 10 μg / mL to about 50 μg / mL. In one embodiment, the concentration of IL12-ABD in the liquid formulation is about 10 μg / mL to about 30 μg / mL. In one embodiment, the concentration of IL12-ABD in the liquid formulation is about 20 μg / mL.

[0068] In one embodiment, the concentration of IL12-ABD in the solid dosage form is about 0.01% to about 0.03% (w / w), about 0.015% to about 0.03% (w / w), about 0.02% to about 0.03% (w / w), about 0.024% to about 0.028% (w / w), about 0.01% to about 0.015% (w / w), about 0.015% to about 0.02% (w / w), about 0.02% to about 0.022% (w / w), about 0.022% to about 0.024% (w / w). 0.024% to about 0.026% (w / w), about 0.026% to about 0.028% (w / w), about 0.028% to about 0.03% (w / w), about 0.01% (w / w), about 0.015% (w / w), about 0.02% (w / w), about 0.022% (w / w), about 0.024% (w / w), about 0.025% (w / w), about 0.026% (w / w), about 0.028% (w / w) and / or about 0.03% (w / w).

[0069] In one embodiment, the concentration of IL12-ABD in the solid formulation is about 0.01% to about 0.03% (w / w). In one embodiment, the concentration of IL12-ABD in the solid formulation is about 0.024% to about 0.028% (w / w). In one embodiment, the concentration of IL12-ABD in the solid formulation is about 0.026% (w / w).

[0070] In one embodiment, the amount of IL12-ABD in the liquid or solid formulation is at least 10 μg, e.g., at least 20 μg, at least 30 μg, at least 40 μg, at least 50 μg, at least 60 μg, at least 80 μg, at least 90 μg, at least 100 μg, at least 110 μg, at least 120 μg, at least 130 μg, at least 140 μg, at least 150 μg, at least 160 μg, at least 180 μg or at least 200 μg. In one embodiment, the amount of IL12-ABD in the liquid or solid formulation is from about 10 μg to about 200 μg, from about 20 μg to about 180 μg, from about 30 μg to about 160 μg, from about 40 μg to about 150 μg, from about 50 μg to about 140 μg, from about 60 μg to about 130 μg, from about 80 μg to about 120 μg, from about 90 μg to about 110 μg, from about 10 μg to about 20 μg, from about 20 μg to about 30 μg, from about 30 μg to about 40 μg, from about 40 μg to about 50 μg, from about 50 μg to about 60 μg, from about 60 μg to about 70 μg, from about 70 μg to about 80 μg, from about 80 μg to about 90 μg, from about 90 μg to about 110 μg. 180 μg, about 180 μg to about 200 μg, about 10 μg, about 20 μg, about 30 μg, about 40 μg, about 50 μg, about 60 μg, about 70 μg, about 80 μg, about 90 μg, about 100 μg, about 110 μg, about 120 μg, about 130 μg, about 140 μg, about 150 μg, about 160 μg, about 180 μg and / or about 200 μg.

[0071] 1.3.2. Chelating agents

[0072] In one embodiment, the formulation comprises a chelating agent.

[0073] In one embodiment, the concentration of the chelating agent in the liquid formulation is about 5 μM to about 20 μM, about 5 μM to about 15 μM, about 5 μM to about 10 μM, about 5 μM to about 10 μM, about 10 μM to about 15 μM, about 15 μM to about 20 μM, about 5 μM, about 10 μM, about 15 μM and / or about 20 μM, preferably about 10 μM.

[0074] In one embodiment, the concentration of the chelating agent in the solid formulation is from about 0.002% to about 0.006% (w / w), from about 0.003% to about 0.006% (w / w), from about 0.004% to about 0.006% (w / w), from about 0.0045% to about 0.0055% (w / w), from about 0.002% to about 0.003% (w / w), from about 0.003% to about 0.004% (w / w), from about 0.0045% to about 0.0055% (w / w), from about 0.002% to about 0.003% (w / w), from about 0.003% to about 0.004% (w / w), from about 0.004% to about 0.005% (w / w), about 0.005% to about 0.006% (w / w), about 0.002% (w / w), about 0.003% (w / w), about 0.004% (w / w), about 0.0045% (w / w), about 0.005% (w / w), about 0.0055% (w / w) and / or about 0.0066% (w / w), preferably about 0.005% (w / w).

[0075] In one embodiment, the chelating agent is a naturally occurring compound. In one embodiment, the chelating agent is a synthetic compound. In one embodiment, the chelating agent is diethylenetriaminepentaacetic acid (DTPA).

[0076] 1.3.3. Surfactants

[0077] In one embodiment, the formulation comprises a surfactant.

[0078] Surfactants can reduce the surface tension of a liquid. In one embodiment, the surfactant is a nonionic surfactant. Examples of surfactants include polysorbates (polyoxyethylene sorbitan monolaurate, such as polysorbate 20 and polysorbate 80); TRITON (tert-octylphenoxypolyethoxyethanol); sodium dodecyl sulfate (SDS); sodium lauryl sulfate; sodium octyl glucoside; dodecyl-, myristyl-, linoleyl-, or stearyl-sulfobetaine; dodecyl-, myristyl-, linoleyl-, or stearyl-sarcosine; linoleyl-, myristyl-, linoleyl-, or stearyl-sultaine; Myristyl- or hexadecyl-betaine; lauroyl-, cocamidopropyl-, linoleyl-, myristamidopropyl-, palmitamidopropyl- or isostearamidopropyl-betaine (e.g., lauroyl); myristamidopropyl-, palmitamidopropyl- or isostearamidopropyl-dimethylamine; sodium methyl cocoyl-taurate or disodium methyl oleyl-taurate; sorbitan monopalmitate; MONAQUAT series (Mona Industries, Inc., Paterson, NJ); polyethylene glycol (PEG), polypropylene glycol (PPG) and copolymers of polyoxyethylene and polyoxypropylene glycol (e.g., Pluronics / Poloxamer, PF68, etc.). In one embodiment, the surfactant is a polysorbate. In one embodiment, the polysorbate is polysorbate 20, polysorbate 40, polysorbate 60 or polysorbate 80.

[0079] In one embodiment, the liquid formulation comprises a surfactant at a concentration of about 0.01% to about 0.04%, about 0.01% to about 0.03%, about 0.01% to about 0.02%, about 0.02% to about 0.03%, about 0.03% to about 0.04%, about 0.01%, about 0.02%, about 0.03% and / or about 0.04% (w / v), preferably about 0.02% (w / v).

[0080] In one embodiment, the solid formulation comprises a surfactant at a concentration of about 0.1% to about 0.4%, about 0.15% to about 0.3%, about 0.1% to about 0.15%, about 0.15% to about 0.2%, about 0.2% to about 0.3%, about 0.3% to about 0.4%, about 0.1%, about 0.15%, about 0.2%, about 0.3% and / or about 0.4% (w / w), preferably about 0.2% (w / w).

[0081] 1.3.4. Polysorbates

[0082] In one embodiment, the formulation comprises polysorbate.

[0083] 1.3.4.1. Polysorbate 80

[0084] In one embodiment, the formulation comprises polysorbate 80 (PS80).

[0085] In one embodiment, the liquid formulation comprises about 0.01% to about 0.04%, about 0.01% to about 0.03%, about 0.01% to about 0.02%, about 0.02% to about 0.03%, about 0.03% to about 0.04%, about 0.01%, about 0.02%, about 0.03%, and / or about 0.04% (w / v) polysorbate 80. In one embodiment, the formulation comprises about 0.02% (w / v) polysorbate 80.

[0086] In one embodiment, the solid formulation comprises about 0.1% to about 0.4%, about 0.15% to about 0.3%, about 0.1% to about 0.15%, about 0.15% to about 0.2%, about 0.2% to about 0.3%, about 0.3% to about 0.4%, about 0.1%, about 0.15%, about 0.2%, about 0.3%, and / or about 0.4% (w / w) polysorbate 80. In one embodiment, the formulation comprises about 0.2% (w / w) polysorbate 80.

[0087] 1.3.4.2. Polysorbate 60

[0088] In one embodiment, the formulation comprises polysorbate 60 (PS60).

[0089] In one embodiment, the liquid formulation comprises about 0.01% to about 0.04%, about 0.01% to about 0.03%, about 0.01% to about 0.02%, about 0.02% to about 0.03%, about 0.03% to about 0.04%, about 0.01%, about 0.02%, about 0.03%, and / or about 0.04% (w / v) polysorbate 60. In one embodiment, the formulation comprises about 0.02% (w / v) polysorbate 60.

[0090] In one embodiment, the solid formulation comprises about 0.1% to about 0.4%, about 0.15% to about 0.3%, about 0.1% to about 0.15%, about 0.15% to about 0.2%, about 0.2% to about 0.3%, about 0.3% to about 0.4%, about 0.1%, about 0.15%, about 0.2%, about 0.3%, and / or about 0.4% (w / w) polysorbate 60. In one embodiment, the formulation comprises about 0.2% (w / w) polysorbate 60.

[0091] 1.3.4.3. Polysorbate 40

[0092] In one embodiment, the formulation comprises polysorbate 40 (PS40).

[0093] In one embodiment, the liquid formulation comprises about 0.01% to about 0.04%, about 0.01% to about 0.03%, about 0.01% to about 0.02%, about 0.02% to about 0.03%, about 0.03% to about 0.04%, about 0.01%, about 0.02%, about 0.03%, and / or about 0.04% (w / v) polysorbate 40. In one embodiment, the formulation comprises about 0.02% (w / v) polysorbate 40.

[0094] In one embodiment, the solid formulation comprises about 0.1% to about 0.4%, about 0.15% to about 0.3%, about 0.1% to about 0.15%, about 0.15% to about 0.2%, about 0.2% to about 0.3%, about 0.3% to about 0.4%, about 0.1%, about 0.15%, about 0.2%, about 0.3%, and / or about 0.4% (w / w) polysorbate 40. In one embodiment, the formulation comprises about 0.2% (w / w) polysorbate 40.

[0095] 1.3.4.4. Polysorbate 20

[0096] In one embodiment, the formulation comprises polysorbate 20 (PS20).

[0097] In one embodiment, the liquid formulation comprises about 0.01% to about 0.04%, about 0.01% to about 0.03%, about 0.01% to about 0.02%, about 0.02% to about 0.03%, about 0.03% to about 0.04%, about 0.01%, about 0.02%, about 0.03%, and / or about 0.04% (v / v) polysorbate 20. In one embodiment, the formulation comprises about 0.02% (v / v) polysorbate 20.

[0098] In one embodiment, the solid formulation comprises about 0.1% to about 0.4%, about 0.15% to about 0.3%, about 0.1% to about 0.15%, about 0.15% to about 0.2%, about 0.2% to about 0.3%, about 0.3% to about 0.4%, about 0.1%, about 0.15%, about 0.2%, about 0.3%, and / or about 0.4% (w / w) polysorbate 20. In one embodiment, the formulation comprises about 0.2% (w / w) polysorbate 20.

[0099] 1.3.5. Sugar

[0100] In one embodiment, liquid preparation comprises sugar and its derivatives, including monosaccharide, disaccharide, trisaccharide, polysaccharide, sugar alcohol, reducing sugar, non-reducing sugar etc. The example of sugar includes glucose, mannose, trehalose, lactose, fructose, maltose, dextran, dextrin, erythritol, glycerol, arabitol, xylitol, sorbitol, mannitol, melibiose, melezitose, raffinose, mannotriose, stachyose, maltose, lactulose, maltulose, glucitol (glucitol), maltitol, lactitol, isomaltulose etc. In one embodiment, sugar is disaccharide. In one embodiment, disaccharide is trehalose. In one embodiment, the concentration of disaccharide is about 1 to about 100mg / mL.

[0101] 1.3.5.1. Trehalose

[0102] In one embodiment, the formulation comprises trehalose.

[0103] In one embodiment, the liquid formulation comprises about 100mM to about 400mM, about 100mM to about 300mM, about 150mM to about 250mM, about 100mM to about 150mM, about 150mM to about 200mM, about 200mM to about 250mM, about 250mM to about 300mM, about 300mM to about 350mM, about 350mM to about 400mM, about 100mM, about 150mM, about 200mM, about 250mM, about 300mM, about 350mM and / or about 400mM trehalose. In one embodiment, the liquid formulation comprises about 200mM trehalose.

[0104] In one embodiment, the solid formulation comprises about 50% to about 95% trehalose, about 60% to about 95% trehalose, about 70% to about 95% trehalose, about 80% to about 95% trehalose, about 85% to about 90% trehalose, about 50% to about 60% trehalose, about 60% to about 70% trehalose, about 70% to about 80% trehalose, about 80% to about 85% trehalose, about 85% to about 90% trehalose, about 90% to about 95% trehalose, about 50% trehalose, about 60% trehalose, about 70% trehalose, about 80% trehalose, about 85% trehalose, about 90% to about 95% trehalose, about 50% trehalose, about 60% trehalose, about 70% trehalose, about 80% trehalose, about 85% trehalose, about 90% trehalose, about 95% trehalose (w / w). In one embodiment, the solid formulation comprises about 70% to about 80% trehalose (w / w). In one embodiment, the solid formulation comprises about 75% trehalose (w / w).

[0105] 1.3.6. Free amino acids

[0106] In one embodiment, the preparation comprises at least one free amino acid. In one embodiment, the preparation comprises only one free amino acid. In one embodiment, the preparation comprises only two free amino acids. In one embodiment, the preparation comprises only three free amino acids. The free amino acids can be L-type, D-type or a mixture of these forms.

[0107] In one embodiment, at least one free amino acid is glycine, alanine, valine, leucine, isoleucine, proline, serine, threonine, glutamine, asparagine, cysteine, methionine, phenylalanine, tyrosine, tryptophan, aspartic acid, glutamic acid, histidine, arginine or lysine. In one embodiment, at least one free amino acid is glycine, alanine, proline, serine, aspartic acid, arginine or lysine.

[0108] In one embodiment, the concentration of the free amino acid in the liquid preparation is about 25mM to about 100mM, about 25mM to about 75mM, about 40mM to about 60mM, about 25mM to about 30mM, about 30mM to about 40mM, about 40mM to about 50mM, about 50mM to about 60mM, about 60mM to about 70mM, about 70mM to about 80mM, about 80mM to about 90mM, about 90mM to about 100mM, about 25mM, about 30mM, about 40mM, about 50mM, about 60mM, about 70mM, about 80mM, about 90mM and / or about 100mM. In one embodiment, the concentration of the free amino acid in the liquid preparation is about 50mM.

[0109] In one embodiment, the concentration of free amino acids in the solid dosage form is about 2% to about 8%, about 3% to about 7%, about 3% to about 6%, about 4% to about 5%, about 4.2% to about 4.8%, about 4.4% to about 4.6%, about 2% to about 3%, about 3% to about 4%, about 4% to about 4.1%, about 4.1% to about 4.2%, about 4.2% to about 4.3%, about 4.3% to about 4.4%, about 4.4% to about 4.5%, about 4.6% to about 4.7%, about 4.8% to about 4.9%, about 4.9% to about 4.10 ...6% to about 4.7%, about 4.8% to about 4.9%, about 4.9% to about 4.10%, about 4.6% to about 4.7%, about 4.8% to about 4.9%, about 4.9% to about 4.10%, about 4.6% to about 4.9%, about About 4.5% to about 4.6%, about 4.6% to about 4.7%, about 4.7% to about 4.8%, about 4.8% to about 4.9%, about 4.9% to about 5%, about 2%, about 3%, about 4%, about 4.1%, about 4.2%, about 4.3%, about 4.4%, about 4.45%, about 4.5%, about 4.6%, about 4.7%, about 4.8%, about 4.9%, about 5%, about 6%, about 7% and / or about 8% (w / w). In one embodiment, the concentration of the free amino acids in the solid preparation is about 4% and about 5% (w / w). In one embodiment, the concentration of the free amino acids in the solid preparation is about 4.45% (w / w).

[0110] In one embodiment, the formulation comprises alanine and at least one additional free amino acid selected from glycine, proline, serine, aspartic acid, arginine and lysine. In one embodiment, the formulation comprises alanine and arginine.

[0111] 1.3.6.1. Alanine

[0112] In one embodiment, the formulation comprises alanine.

[0113] In one embodiment, the liquid formulation comprises about 25mM to about 100mM, about 25mM to about 75mM, about 40mM to about 60mM, about 25mM to about 30mM, about 30mM to about 40mM, about 40mM to about 50mM, about 50mM to about 60mM, about 60mM to about 70mM, about 70mM to about 80mM, about 80mM to about 90mM, about 90mM to about 100mM, about 25mM, about 30mM, about 40mM, about 50mM, about 60mM, about 70mM, about 80mM, about 90mM and / or about 100mM alanine. In one embodiment, the liquid formulation comprises about 50mM alanine.

[0114] In one embodiment, the solid dosage form comprises about 2% to about 8%, about 3% to about 7%, about 3% to about 6%, about 4% to about 5%, about 4.2% to about 4.8%, about 4.4% to about 4.6%, about 2% to about 3%, about 3% to about 4%, about 4% to about 4.1%, about 4.1% to about 4.2%, about 4.2% to about 4.3%, about 4.3% to about 4.4%, about 4.4% to about 4.5%, about 4.5% to about 4.6%. In one embodiment, the solid formulation comprises about 4% and about 5% alanine (w / w). In one embodiment, the solid formulation comprises about 4.45% alanine (w / w).

[0115] 1.3.6.2. Arginine

[0116] In one embodiment, the formulation comprises arginine.

[0117] In one embodiment, the liquid preparation comprises about 25mM to about 100mM, about 25mM to about 75mM, about 40mM to about 60mM, about 25mM to about 30mM, about 30mM to about 40mM, about 40mM to about 50mM, about 50mM to about 60mM, about 60mM to about 70mM, about 70mM to about 80mM, about 80mM to about 90mM, about 90mM to about 100mM, about 25mM, about 30mM, about 40mM, about 50mM, about 60mM, about 70mM, about 80mM, about 90mM and / or about 100mM arginine. In one embodiment, the liquid preparation comprises about 50mM arginine.

[0118] In one embodiment, the solid dosage form comprises about 2% to about 8%, about 3% to about 7%, about 3% to about 6%, about 4% to about 5%, about 4.2% to about 4.8%, about 4.4% to about 4.6%, about 2% to about 3%, about 3% to about 4%, about 4% to about 4.1%, about 4.1% to about 4.2%, about 4.2% to about 4.3%, about 4.3% to about 4.4%, about 4.4% to about 4.5%, about 4.5% to about 4.6%. In one embodiment, the solid formulation comprises about 4% and about 5% arginine (w / w). In one embodiment, the solid formulation comprises about 4.45% arginine (w / w).

[0119] 1.3.7. Buffer

[0120] In one embodiment, the formulation comprises at least one buffer (buffer component). Typically, the buffer (when present) is used to adjust the pH of the formulation to about 6.8 to about 8.0, about 7.0 to about 7.8, about 7.2 to about 7.8, about 7.4 to about 7.6, about 6.8 to about 7.0, about 7.0 to about 7.2, about 7.2 to about 7.4, about 7.4 to about 7.6, about 7.6 to about 7.8, about 7.8 to about 8.0, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9 and / or about 8.0.

[0121] In one embodiment, at least one buffer is selected from acetate, succinate, gluconate, histidine, citrate, phosphate, maleate, cacodylate, tris (hydroxymethyl) aminomethane (Tris), 2-[N-morpholino] ethanesulfonic acid (MES), bis (2-hydroxyethyl) iminotris [hydroxymethyl] methane (Bis-Tris), N- [2-acetylamino] -2-iminodiacetic acid (ADA), glycylglycine and other organic acid buffers. In one embodiment, the buffer is glycylglycine, Tris, histidine, citrate, phosphate, succinate or acetate.

[0122] In one embodiment, the concentration of the buffer component in the liquid formulation is about 10mM to about 50mM, about 10mM to about 40mM, about 10mM to about 30mM, about 10mM to about 15mM, about 15mM to about 20mM, about 20mM to about 25mM, about 25mM to about 30mM, about 35mM to about 40mM, about 40mM to about 45mM, about 45mM to about 50mM, about 10mM, about 15mM, about 20mM, about 25mM, about 30mM, about 35mM, about 40mM, about 45mM to about 50mM, about 10mM, about 15mM, about 20mM, about 25mM, about 30mM, about 35mM, about 40mM, about 45mM and / or about 50mM. In one embodiment, the concentration of the buffer component in the liquid formulation is about 25mM.

[0123] In one embodiment, the concentration of the buffer component in the solid dosage form is about 2% to about 8%, about 3% to about 7%, about 3% to about 6%, about 3% to about 4%, about 3.2% to about 3.6%, about 2% to about 3%, about 3% to about 3.1%, about 3.1% to about 3.2%, about 3.2% to about 3.3%, about 3.3% to about 3.4%, about 3.4% to about 3.5%, about 3.5% to about 3.6%, about 3.6% to about 3.7%. %, about 3.7% to about 3.8%, about 3.8% to about 3.9%, about 3.9% to about 4%, about 4% to about 5%, about 5% to about 6%, about 6% to about 7%, about 7% to about 8%, about 2%, about 3%, about 3.1%, about 3.2%, about 3.3%, about 3.4%, about 3.5%, about 3.6%, about 3.7%, about 3.8%, about 3.9%, about 4%, about 5%, about 6%, about 7% and / or about 8% (w / w). In one embodiment, the concentration of the buffer component in the solid dosage form is about 3% to about 4% (w / w). In one embodiment, the concentration of the buffer component in the solid dosage form is about 3.4% (w / w).

[0124] In one embodiment, the buffer is glycylglycine. In one embodiment, the formulation comprises glycylglycine.

[0125] In one embodiment, the liquid formulation comprises about 10mM to about 40mM, about 10mM to about 30mM, about 10mM to about 15mM, about 15mM to about 20mM, about 20mM to about 25mM, about 25mM to about 30mM, about 35mM to about 40mM, about 40mM to about 45mM, about 45mM to about 50mM, about 10mM, about 15mM, about 20mM, about 25mM, about 30mM, about 35mM, about 40mM, about 45mM and / or about 50mM glycyl glycine. In one embodiment, the liquid formulation comprises about 25mM glycyl glycine.

[0126] In one embodiment, the solid dosage form comprises about 2% to about 8%, about 3% to about 7%, about 3% to about 6%, about 3% to about 4%, about 3.2% to about 3.6%, about 2% to about 3%, about 3% to about 3.1%, about 3.1% to about 3.2%, about 3.2% to about 3.3%, about 3.3% to about 3.4%, about 3.4% to about 3.5%, about 3.5% to about 3.6%, about 3.6% to about 3.7%, about 3.7% to about 3. About 3.8%, about 3.8% to about 3.9%, about 3.9% to about 4%, about 4% to about 5%, about 5% to about 6%, about 6% to about 7%, about 7% to about 8%, about 2%, about 3%, about 3.1%, about 3.2%, about 3.3%, about 3.4%, about 3.5%, about 3.6%, about 3.7%, about 3.8%, about 3.9%, about 4%, about 5%, about 6%, about 7% and / or about 8% glycylglycine (w / w). In one embodiment, the solid preparation comprises about 3% to about 4% glycylglycine (w / w). In one embodiment, the solid preparation comprises about 3.4% glycylglycine (w / w).

[0127] 1.3.7.1.pH

[0128] In one embodiment, the pH of the liquid formulation is about 6.8 to about 8.0, such as about 7.0 to about 7.8, about 7.2 to about 7.8, about 7.4 to about 7.6, about 6.8 to about 7.0, about 7.0 to about 7.2, about 7.2 to about 7.4, about 7.4 to about 7.6, about 7.6 to about 7.8, about 7.8 to about 8.0, about 6.8, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9 and / or about 8.0. In one embodiment, the pH of the formulation is about 7.5.

[0129] 1.3.8. Stabilization

[0130] In one embodiment, the liquid formulation stabilizes the IL12-ABD fusion protein under thermal stress. In one embodiment, the liquid formulation reduces the formation of sub-visible particles measured by FlowCam after the thermal stress at 40°C continues for fourteen days. In one embodiment, the liquid formulation reduces the formation of high molecular weight (HMW) substances measured by size exclusion high performance liquid chromatography (SEC-HPLC) after the thermal stress at 40°C continues for fourteen days. In one embodiment, the liquid formulation reduces the formation of low molecular weight (LMW) substances measured by size exclusion high performance liquid chromatography (SEC-HPLC) after the thermal stress at 40°C continues for fourteen days.

[0131] In one embodiment, liquid preparations stabilize IL12-ABD fusion protein under freeze-thaw stress. In one embodiment, liquid preparations reduce the formation of sub-visible particles measured by FlowCam after the freeze-thaw stress of five freeze-thaw cycles. In one embodiment, liquid preparations reduce the formation of high molecular weight (HMW) substances measured by size exclusion high performance liquid chromatography (SEC-HPLC) after the freeze-thaw stress of five freeze-thaw cycles. In one embodiment, liquid preparations reduce the formation of low molecular weight (LMW) substances measured by size exclusion high performance liquid chromatography (SEC-HPLC) after the freeze-thaw stress of five freeze-thaw cycles.

[0132] In one embodiment, liquid preparations stabilize IL12-ABD fusion protein under shear stress. In one embodiment, liquid preparations reduce the formation of sub-visible particles measured by FlowCam after the simulated shear stress of 900RPM continues for 24 hours. In one embodiment, liquid preparations reduce the formation of high molecular weight (HMW) substances measured by size exclusion high performance liquid chromatography (SEC-HPLC) after the simulated shear stress of 900RPM continues for 24 hours. In one embodiment, liquid preparations reduce the formation of low molecular weight (LMW) substances measured by size exclusion high performance liquid chromatography (SEC-HPLC) after the simulated shear stress of 900RPM continues for 24 hours.

[0133] 1.4. Administration of the preparation

[0134] Suitable routes of administration of the IL12-ABD fusion protein formulations described herein include, but are not limited to, parenteral administration (e.g., by subcutaneous, intravenous, intramuscular, intradermal, or intrasternal injection or infusion (e.g., as a sterile injectable aqueous or non-aqueous solution or suspension, etc.)). In certain embodiments, the formulation is suitable for parenteral injection. In certain embodiments, the formulation is suitable for intravenous injection. In certain embodiments, the formulation is suitable for subcutaneous injection.

[0135] In some embodiments, the formulation is suitable for single or multiple dose administration.

[0136] 1.5. Dosage form

[0137] In another aspect, provided herein is a dosage form containing one or more unit doses of a pharmaceutical composition comprising an IL12-ABD fusion protein.

[0138] In various embodiments, the dosage form is a prefilled syringe. In various embodiments, the dosage form is an autoinjector pen.

[0139] In various embodiments, the dosage form comprises the above-mentioned preparation of one or more unit doses. In typical embodiments, the preparation is a liquid preparation. In other embodiments, the preparation is a dry preparation, including but not limited to lyophilized material. In a special embodiment, the dosage form comprises a dry preparation and a quantitative aqueous diluent.

[0140] In some embodiments, the unit dosage form comprises a formulation comprising about 10 μg / mL to about 50 μg / mL of an IL12-ABD fusion protein, about 25 mM to about 100 mM alanine, about 100 mM to about 400 mM trehalose, about 10 mM to about 50 mM glycylglycine, about 0.01% to about 0.04% polysorbate 20 (v / v), and about 5 μM to about 20 μM diethylenetriaminepentaacetic acid (DTPA), wherein the pH of the formulation is about 6.8 to about 8.0.

[0141] In some embodiments, the unit dosage form comprises a formulation comprising about 0.01% to about 0.03% (w / w) IL12-ABD fusion protein, about 2% to about 8% alanine (w / w), about 50% to about 95% trehalose (w / w), about 2% to about 8% glycylglycine (w / w), about 0.1% to about 0.4% polysorbate 20 (w / w), and about 0.002% to about 0.006% diethylenetriaminepentaacetic acid (DTPA) (w / w).

[0142] 1.6. Treatment methods

[0143] In another aspect, provided herein is a method for treating a disease or condition in a patient, comprising administering to the patient an IL12-ABD fusion protein formulation as described herein. In some embodiments, the patient suffers from an IL-12 related disease or condition, such as cancer, an autoimmune disease, or an infectious disease. In some embodiments, the IL12-ABD fusion protein is administered as an immunoadjuvant for the treatment of cancer, an autoimmune disease, or an infectious disease, such as by administering the IL12-ABD fusion protein together with a vaccine antigen.

[0144] In another aspect, provided herein is a method of inhibiting tumor growth in a patient, comprising administering to the patient an IL12-ABD fusion protein formulation described herein.

[0145] In another aspect, provided herein are methods of treating cancer in a patient comprising administering to the patient an IL12-ABD fusion protein formulation described herein.

[0146] In one embodiment, the cancer to be treated includes, but is not limited to, carcinoma, blastoma, sarcoma, and certain leukemias or lymphoid malignancies. More specific examples of such cancers include squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous carcinoma), peritoneal cancer, hepatocellular carcinoma, gastric cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, skin cancer / melanoma, and head and neck cancer and metastases associated with any primary tumor.

[0147] In another aspect, provided herein is a method of treating an autoimmune disease in a patient comprising administering to the patient an IL12-ABD fusion protein formulation described herein.

[0148] In one embodiment, autoimmune diseases to be treated include, but are not limited to, rheumatoid arthritis, juvenile idiopathic arthritis, and graft versus host disease (GvHD).

[0149] In another aspect, provided herein is a method of treating an infectious disease in a patient comprising administering to the patient an IL12-ABD fusion protein formulation described herein.

[0150] In one embodiment, the infectious diseases to be treated include, but are not limited to, local or systemic viral infections, such as encephalitis, influenza, the common cold, immunodeficiency, and herpes virus infections. In one embodiment, the infectious diseases are caused by bacteria, viruses, protozoa, helminths, or other microbial pathogens. In one embodiment, the infections to be treated are caused by hepatitis viruses, human immunodeficiency virus (HIV), human T-lymphotropic virus (HTLV), herpes viruses, Epstein-Barr virus, or human papillomavirus.

[0151] The following are examples of specific embodiments of the present invention. These examples are for illustrative purposes only and are not intended to limit the scope of the present invention in any way. Every effort has been made to ensure the accuracy of the numbers used (e.g., amounts, temperatures, etc.), but of course some experimental errors and deviations should be allowed.

[0152] Example

[0153] Example 1 : Stability study of formulations with different buffers and pH

[0154] The preparation of IL12-ABD fusion protein SONE-IL12-ABD (SEQ ID NO: 11) was prepared as shown in Table 1.

[0155] Table 1:

[0156]

[0157] 1.1. Thermal stress study

[0158] The preparations were subjected to heat stress at 40°C for 7 days and monitored for osmotic pressure, pH, A280, right angle light scattering (RALS), DLS, Flowcam, SEC-HPLC and reduced and non-reduced SDS-page. The results of osmotic pressure, pH, A280 and RALS are shown in Table 2.

[0159] Table 2:

[0160]

[0161]

[0162] As shown in Table 2, the concentrations of formulations containing acetate and citrate increased the most after 7 days of incubation at 40°C. In addition, as assessed using RALS, formulations with higher pH and formulations containing citrate (having three carboxyl groups) had fewer light scattering particles (i.e., lower RALS values) after heat stress compared to formulations containing acetate (having one carboxyl group) and formulations containing succinate (having two carboxyl groups).

[0163] The formulations were evaluated by DLS after incubation for 7 days at 40° C. As shown in Table 3, the particle size of formulations with a pH less than 7 increased after thermal stress.

[0164] Table 3:

[0165]

[0166] After incubation at 40°C for 7 days, the preparations were evaluated by Flowcam for the presence of subparticles. The results are shown in Table 4.

[0167] Table 4:

[0168]

[0169] The preparations were subjected to heat stress at 40°C for 7 days, and the total recovery, fusion protein recovery, and impurity percentages consisting of HMW and LMW were monitored by SEC-HPLC. The results are shown in Tables 5 and 6. At T7 after heat stress, preparations with pH <7.0 showed higher HMW material % and lower main recovery %. Preparations with higher pH (>7.0) (A1-A4 and A-CF) were stable to heat stress at T7 with minimal aggregation.

[0170] Table 5:

[0171]

[0172]

[0173] Table 6:

[0174]

[0175] Formulations heat stressed at 40°C for 7 days were also analyzed by reducing and non-reducing SDS-PAGE. Each formulation was consistent with A-CF on the reducing gel. The non-reducing gel showed more variability and additional HMW bands in A5 and A6 with histidine and with and without PS20, A7 and A8 with water and with and without PS20, A9 and A10 with acetate and with and without PS20, and A11 and A12 with citrate and with and without PS20.

[0176] 1.2. Freeze-thaw studies

[0177] The formulation at T0 was subjected to five freeze-thaw cycles and then analyzed for osmotic pressure, pH, RALS, A280, DLS, SEC-HPLC, and reduced and non-reduced SDS-page. The results of osmotic pressure, pH, A280, and right angle light scattering (RALS) are shown in Table 7.

[0178] Table 7:

[0179]

[0180] As shown in Table 7, after freeze-thaw cycles, A7 and A8 containing water with and without PS20, A9 containing acetate and PS20, and A14 containing succinate had lower recoveries with concentration percentages below 90%. In addition, as assessed using RALS, the trend was consistent with temperature stress, with formulations with higher pH and formulations containing citrate having the lowest RALS values.

[0181] After five freeze-thaw cycles, after incubation, the formulations were evaluated by DLS. As shown in Table 8, after five freeze-thaw cycles, the polydispersity index (PDI) of all formulations increased except A-CF.

[0182] Table 8:

[0183]

[0184]

[0185] The formulations were subjected to five freeze-thaw cycles and the total recovery, fusion protein recovery, and percent impurities consisting of HMW and LMW were monitored by SEC-HPLC. The results are shown in Tables 9 and 10. The formulations were sensitive to freeze-thaw. Consistent with the effects of time and temperature (thermal stress studies), the % recovery of the main and total peaks for the formulations with higher pH was relatively high, and the increase in LMW species was less.

[0186] Table 9:

[0187]

[0188] Table 10:

[0189]

[0190] The preparations that had been subjected to five freeze-thaw cycles were also analyzed by reducing and non-reducing SDS-PAGE. Each preparation was consistent with A-CF on both reducing and non-reducing gels.

[0191] 1.3. Simulated shear stress study

[0192] The formulations at T0 were subjected to simulated shear stress at 900 RPM for 24 hours at room temperature and analyzed for osmotic pressure, pH, RALS, DLS, SEC and SDS-PAGE. The results of osmotic pressure, pH and RALS are shown in Table 11.

[0193] Table 11:

[0194]

[0195]

[0196] *Insufficient samples

[0197] As shown in Table 11, consistent with the results obtained for thermal stress and freeze-thaw stress, formulations with a pH above pH 7.0 exhibited relatively less light scattering after simulated shear stress. The data also show that the SONE-IL12-ABD formulation is susceptible to simulated shear stress. Except for the formulation containing phosphate and no PS20 at pH 7.0, all formulations with a pH value above 6.0 remained transparent under simulated shear stress.

[0198] After incubation after simulated shear stress, the formulations were evaluated by DLS. As shown in Table 12, after simulated shear stress, the particle size of all formulations increased significantly, while the particle size of the formulation with pH>7.0 was relatively small compared to the other formulations.

[0199] Table 12:

[0200]

[0201]

[0202] The preparations were subjected to a simulated shear stress of 900 RPM for 24 hours and the total recovery, fusion protein recovery, and the percentage of impurities consisting of HMW and LMW were monitored by SEC-HPLC. The results are shown in Tables 13 and 14. The aggregation degree of preparation A10 containing no PS20 and acetate and preparation A12 containing citrate was higher. The preparation containing Tris showed the highest recovery after simulated shear stress.

[0203] Table 13:

[0204]

[0205] Table 14:

[0206]

[0207]

[0208] Formulations subjected to simulated shear stress were also analyzed by reducing and non-reducing SDS-PAGE. After shear stress, each formulation was consistent with A-CF on the reducing gel, except for formulation A12 containing citrate. After shear stress, the non-reducing gel showed greater variability, increased degradation in formulation A3 containing Tris and PS20, and HMW streaks in multiple samples.

[0209] The results show that SONE-IL12-ABD formulations containing only buffer and with and without PS20 are sensitive to heat, freeze-thaw and shear. The formulation stability depends on pH, and formulations with a pH equal to or higher than 7.0 show relatively higher stability when analyzed after thermal stress, freeze-thaw stress and simulated shear stress. Formulations containing phosphate and Tris and with and without PS20 are relatively more stable than other formulations under physical and thermal stress. The example of this formulation contains 10mM Tris (pH is 7.5) and 0.02% PS20.

[0210] Example 2 :Stability study of formulations with different ionic strengths

[0211] Formulations of SONE-IL12-ABD were prepared as shown in Table 15 using a SONE-IL12-ABD concentration of about 0.050 mg / ml.

[0212] Table 15:

[0213]

[0214] The formulations were subjected to temperature ramping RALS and IF to obtain the Tagg and Tm of each formulation. The Tm and Tagg values ​​are shown in Table 16.

[0215] Table 16:

[0216]

[0217] The samples with high salt and B-CF had the highest Tm. The samples without salt (B1 without NaCl, B6 with Tris, B7 with histidine, B-CF) had the highest Tagg.

[0218] Thermal stress studies

[0219] The formulations were heat stressed at 40°C for 14 days and monitored for osmotic pressure, pH, A280, RALS, Flowcam, SEC-HPLC, and reduced and non-reduced SDS-page. The results of osmotic pressure, pH, A280, and RALS are shown in Tables 17 and 18.

[0220] Table 17:

[0221]

[0222] Table 18:

[0223]

[0224]

[0225] As shown in Table 18, for formulations B2 and B3 containing increasing amounts of salt, the percent recovery at T14 was lower. Formulation B5 containing high salt had high recoveries, as did formulations B6 and B-CF containing Tris. B1 without salt, B2 containing 10 mM NaCl, and B7 containing histidine had recoveries ranging from 85% to 105%.

[0226] After 7 and 14 days of incubation at 40°C, the formulations were evaluated by Flowcam for the presence of subparticles. The results are shown in Table 19.

[0227] Table 19:

[0228]

[0229] The preparations were subjected to heat stress treatment at 40°C for 14 days and the total recovery, fusion protein recovery and the percentage of impurities consisting of HMW and LMW were monitored by SEC-HPLC. The results are shown in Tables 20 to 22. B3 containing 50 mM NaCl had the lowest amount of %LMW at T14. Preparations containing Tris and histidine (B6 and B7) had significantly increased HMW and LMW. The preparation containing histidine (B7) had the highest total recovery at T14.

[0230] Table 20:

[0231]

[0232]

[0233] Table 21:

[0234]

[0235] Table 22:

[0236]

[0237] The formulations heat stressed at 40°C for 14 days were also analyzed by reducing and non-reducing SDS-PAGE. In the non-reducing gel, B1 without salt, B2 with 10 mM NaCl, B6 with Tris, and B7 with histidine had large HMW bands. B1 without salt, B6 with Tris, B7 with histidine, and B-CF also had higher concentrations of LMW bands.

[0238] 2.2. Freeze-thaw studies

[0239] The formulations at T0 were subjected to five freeze-thaw cycles and then analyzed for osmotic pressure, pH, RALS, A280, Flowcam, SEC-HPLC, and reduced and non-reduced SDS-page. The results of osmotic pressure, pH, A280, and RALS are shown in Table 23.

[0240] Table 23:

[0241]

[0242] As shown in Table 23, after freeze-thaw cycles, formulation B3 containing 50 mM NaCl had low concentration recovery after freeze-thaw. B5 containing high salt, B6 containing Tris, and B-CF had recoveries greater than 100%. In addition, formulation B6 containing Tris had a slight increase in RALS values ​​after freeze-thaw.

[0243] After five freeze-thaw cycles, the formulations were evaluated for the presence of subparticles by Flowcam. The results are shown in Table 24.

[0244] Table 24:

[0245]

[0246]

[0247] The preparations were subjected to five freeze-thaw cycles and total recovery, fusion protein recovery, and impurity percentages consisting of HMW and LMW were monitored by SEC-HPLC. The results are shown in Tables 25 and 26. The preparation B5 containing high salt had no LMW after freeze-thaw. B6 containing Tris, B7 containing histidine, and B-CF without salt had the highest total recovery after freeze-thaw.

[0248] Table 25:

[0249]

[0250] Table 26:

[0251]

[0252] The preparations subjected to five freeze-thaw cycles were analyzed by reducing and non-reducing SDS-PAGE. After five freeze-thaw cycles, no significant differences were observed by SDS-PAGE.

[0253] 2.3 Simulated shear stress study

[0254] The formulation at T0 was subjected to simulated shear stress of 900RPM at room temperature for 24 hours and analyzed for osmotic pressure, pH, RALS, DLS, SEC and SDS-PAGE. The results of osmotic pressure, pH and RALS are shown in Table 27.

[0255] Table 27:

[0256]

[0257] As shown in Table 27, the RALS values ​​increased for all formulations, especially the formulations containing 50 mM NaCl, 100 mM NaCl and histidine (B3, B4 and B7).

[0258] After incubation after simulated shear stress, the preparations were evaluated by DLS. As shown in Table 28, all preparations had large particle diameters after simulated shear stress. The sample concentration was about 0.050 mg / ml, so dynamic light scattering was not observed at this low concentration, but under shear stress, the particle size increased, so it can be measured by DLS. This is used as a screening tool for preparations receiving simulated shear stress. Data show that preparations (D1 and D2) containing 0 mM or 10 mM NaCl show low light scattering measured by DLS, and have a smaller particle diameter relative to other preparations.

[0259] Table 28:

[0260]

[0261]

[0262] The preparation is accepted the simulated shear stress of 900RPM for 24 hours, and the total recovery, fusion protein recovery and the impurity percentage consisting of HMW and LMW are monitored by SEC-HPLC.The results are shown in Table 29 and Table 30.Preparation B5 and B-CF containing high salt have the highest % mainly after the simulated shear stress.Compared with other preparations, preparation B5 and CF containing high salt also have very low HMW and LMW.Preparation 1 and CF without salt have the highest total recovery percentage, greater than 90%.

[0263] Table 29:

[0264]

[0265] Table 30:

[0266]

[0267] The formulations subjected to simulated shear stress were also analyzed by reducing and non-reducing SDS-PAGE. Higher aggregation relative to B-CF was observed in the non-reducing gel. Fragmentation was observed in B2 containing 10 mM NaCl and B3 containing 50 mM NaCl.

[0268] The results showed that the SONE-IL12-ABD formulation was more susceptible to simulated shear stress in the presence of NaCl. The lower salt formulations had lower Tm measurements at T0, and the highest Tagg measurements were obtained for the formulation without added salt at pH 7.5. Freeze-thaw studies showed that the sensitivity of each formulation was similar, except for B-CF. In addition, temperature stress studies showed that the formulations without salt had relatively higher total recoveries compared to the formulations containing salt.

[0269] Example 3 :Stability study of formulations with different surfactants

[0270] Formulations of SONE-IL12-ABD were prepared as shown in Table 31 using a SONE-IL12-ABD concentration of about 0.050 mg / ml.

[0271] Table 31:

[0272]

[0273] The formulations were subjected to temperature ramps RALS and IF to obtain the Tagg and Tm of each formulation. The Tm and Tagg values ​​are shown in Table 32.

[0274] Table 32:

[0275]

[0276] Formulation C6 (0.05 mg / ml SONE-IL12-ABD, 25 mM glycylglycine, 0.02% PS20, 10 uM DTP A) exhibited high Tm and Tagg values ​​of 54°C and 70°C, respectively.

[0277] Thermal stress studies

[0278] The formulations were heat stressed at 40°C for 14 days and monitored for osmotic pressure, pH, A280, RALS, Flowcam, SEC-HPLC, and reduced and non-reduced SDS-page. The results of osmotic pressure, pH, A280, and RALS are shown in Tables 33 and 34.

[0279] Table 33:

[0280]

[0281] Table 34:

[0282]

[0283] As shown in Table 33, the pH increased slightly during the 14-day incubation period. Formulations C5 and C-CF containing PEG 3350 had the highest percent recovery based on A280 concentration at T14, approximately 90%.

[0284] The presence of subparticles in the formulations was assessed by Flowcam after 7 and 14 days of incubation at 40° C. As shown in Table 35, at T7 and T14, the subvisible particle counts increased for each formulation, with the largest increase for C4 containing poloxamer 188.

[0285] Table 35:

[0286]

[0287]

[0288] The preparations were subjected to heat stress treatment at 40°C for 14 days, and the total recovery, fusion protein recovery, and the percentage of impurities consisting of HMW and LMW were monitored by SEC-HPLC. The results are shown in Tables 36 to 38. After 14 days of incubation, the %HMW of all preparations increased significantly except C-CF. The total recovery at T14 ranged from 88% of preparation C2 (containing 25mM glycylglycine, 0.02% PS20) to 96% of preparation C6 (containing 25mM glycylglycine, 0.02% PS20 and 10uM DTPA). C-CF had a recovery percentage of 103%.

[0289] Table 36:

[0290]

[0291]

[0292] Table 37:

[0293]

[0294] Table 38:

[0295]

[0296] Formulations heat stressed at 40°C for 14 days were also analyzed by reducing and non-reducing SDS-PAGE. The T0 sample showed no HMW in the non-reducing gel, which showed more variability and increased degradation. C6 (containing PS20 and DTPA) showed 100% major in the non-reducing gel at T14.

[0297] 3.2. Freeze-thaw studies

[0298] The formulations at T0 were subjected to five freeze-thaw cycles and then analyzed for osmotic pressure, pH, RALS, A280, Flowcam, SEC-HPLC, and reduced and non-reduced SDS-page. The results for osmotic pressure, pH, A280, and RALS are shown in Table 39.

[0299] Table 39:

[0300]

[0301] As shown in Table 39, except for C2 (25 mM glycylglycine, 0.02% PS20) (recovery rate 101%), the concentrations decreased slightly after freeze-thaw.

[0302] After five freeze-thaw cycles, the formulations were evaluated by Flowcam for the presence of subparticles. The results are shown in Table 40. After freeze-thaw, subvisible particles increased in each formulation. C6 (25 mM glycylglycine, 0.02% PS20, 10 uM DTPA) had the largest increase in particles.

[0303] Table 40:

[0304]

[0305]

[0306] The preparations were subjected to five freeze-thaws and total recovery, fusion protein recovery and impurity percentages consisting of HMW and LMW were monitored by SEC-HPLC. The results are shown in Table 41 and Table 42. C3 containing PS80, C6 containing PS20 and DTPA and C-CF had 100% recovery on SEC after freeze-thaw. Except for C1 containing glycylglycine, the HMW of all preparations increased slightly. Each preparation had a main peak percentage greater than 94%. C1 containing glycylglycine had a minimum LMW after freeze-thaw.

[0307] Table 41:

[0308]

[0309] Table 42:

[0310]

[0311] The formulations that were subjected to five freeze-thaw cycles were analyzed by reducing and non-reducing SDS-PAGE. After freeze-thaw, the %LMW of each formulation in the reducing gel increased slightly. No changes were observed in the non-reducing gel, which showed 100% of the main peak.

[0312] 3.3. Simulated shear stress study

[0313] The formulation at T0 was subjected to simulated shear stress of 900RPM at room temperature for 24 hours and analyzed for osmotic pressure, pH, A280, RALS, SEC and SDS-PAGE. The results of osmotic pressure, pH, A280 and RALS are shown in Table 43.

[0314] Table 43:

[0315]

[0316] As shown in Table 43, after simulating shear stress, the RALS values ​​of the formulations containing glycylglycine (C1), the formulation containing PS80 (C3) and C-CF increased significantly, indicating that these formulations are relatively more sensitive to shear stress. C6 containing 25mM glycylglycine, 0.02% PS20, 10μM DPTA has the lowest RALS value, indicating that the formulation is relatively insensitive to shear stress. The formulations containing PS20, PS80 and poloxamer (C2, C3 and C4) respectively present a turbid appearance after simulating shear stress.

[0317] The preparation was subjected to a simulated shear stress of 900RPM for 24 hours and the total recovery, fusion protein recovery, and the percentage of impurities consisting of HMW and LMW were monitored by SEC-HPLC. The results are shown in Table 44 and Table 45. The results show that C6 (25mM glycylglycine, 0.02% PS20, 10uM DTPA) has a relatively higher total area recovery compared to other preparations.

[0318] Table 44:

[0319]

[0320]

[0321] Table 45:

[0322]

[0323] Formulations subjected to simulated shear stress were also analyzed by reducing and non-reducing SDS-PAGE. After shearing, the reducing gel did not show any significant differences, but the non-reducing gel showed an increase in HMW. After shearing, C6 (25mM glycylglycine, 0.02% PS20, 10uM DTPA) had the least amount of HMW on the gel.

[0324] The SDS-PAGE and SEC data after thermal stress and the RALS and SDS-PAGE data after simulated shear stress prove that PS20 improves stability. The SEC total recovery data after thermal stress prove that C6 (containing PS20 and DTPA) and C-CF are relatively more stable in the formulation. In addition, the test after freeze-thaw stress proves that C2 containing PS20, C3 containing PS80, C6 containing PS20 and DTPA, and C-CF are relatively more stable in resisting freeze-thaw stress compared with other formulations.

[0325] Example 4 :Stability study of formulations with different amino acid stabilizers

[0326] Formulations of SONE-IL12-ABD were prepared as shown in Table 46 using a SONE-IL12-ABD concentration of approximately 20 μg / ml.

[0327] Table 46:

[0328]

[0329]

[0330] The formulations were subjected to temperature ramps RALS and IF to obtain the Tagg and Tm for each formulation. The Tm and Tagg values ​​are shown in Table 46A.

[0331] Table 46A:

[0332]

[0333] Thermal stress studies

[0334] The formulations were heat stressed at 40°C for 14 days and monitored for osmotic pressure, pH, A280, RALS, Flowcam, SEC-HPLC, and reduced and non-reduced SDS-page. The results for osmotic pressure, pH, A280, and RALS are shown in Tables 47 and 48.

[0335] Table 47:

[0336]

[0337] Table 48:

[0338]

[0339] As shown in Table 48, except for formulation D2 (containing serine), the concentration of each formulation decreased after 14 days of incubation.

[0340] After 7 and 14 days of incubation at 40°C, the presence of subparticles in the formulations was assessed by Flowcam. As shown in Table 49, most formulations had an increase in subvisible particle counts at T14, except for formulation D5 (containing arginine). Formulation D7 (containing proline) had a significant increase in subvisible particle counts at T14.

[0341] Table 49:

[0342]

[0343]

[0344] The preparations were subjected to heat stress treatment at 40°C for 14 days and the total recovery, fusion protein recovery, and the percentage of impurities consisting of HMW and LMW were monitored by SEC-HPLC. The results are shown in Tables 50 to 52. Preparations D5 (containing arginine) and D6 (containing lysine) had the highest percentage of major peaks on SEC. HMW increased at T7 and T14, with less increase in D5 (containing arginine) and D6 (containing lysine).

[0345] Table 50:

[0346]

[0347] Table 51:

[0348]

[0349]

[0350] Table 52:

[0351]

[0352] The formulations heat stressed at 40°C for 14 days were also analyzed by reducing and non-reducing SDS-PAGE. The results are shown in Tables 53 and 54. The T0 sample showed 100% major bands with no HMW or LMW. Each formulation behaved similarly at T7 and T14. Some HMW were observed in the reducing gel, while some LMW were present in the non-reducing gel.

[0353] Table 53:

[0354]

[0355]

[0356] Table 54:

[0357]

[0358] 4.2. Freeze-thaw studies

[0359] The formulation at T0 was subjected to five freeze-thaw cycles and then analyzed for osmotic pressure, pH, RALS, A280, Flowcam, SEC-HPLC, and reduced and non-reduced SDS-page. The results of osmotic pressure, pH, A280 and RALS are shown in Table 55.

[0360] Table 55:

[0361]

[0362]

[0363] As shown in Table 55, after freeze-thaw, the concentrations decreased slightly except for formulations D2 (containing serine) and D7 (containing proline).

[0364] After five freeze-thaw cycles, the formulations were evaluated by Flowcam for the presence of submicron particles. The results are shown in Table 56. Subvisible particles were increased in most formulations.

[0365] Table 56:

[0366]

[0367] The preparations were subjected to five freeze-thaw cycles and monitored by SEC-HPLC for total recovery, fusion protein recovery, and the percentage of impurities consisting of HMW and LMW. The results are shown in Tables 57 and 58. Preparation D7 (containing proline) showed relatively high HMW, indicating that it was sensitive to repeated freeze-thaw cycles. SEC data demonstrated that preparations containing arginine, lysine, serine, alanine, and glycine were relatively insensitive to repeated freeze-thaw cycles, with recoveries approaching 100% at T14.

[0368] Table 57:

[0369]

[0370] Table 58:

[0371]

[0372] Formulations subjected to five freeze-thaw cycles were also analyzed by reducing and non-reducing SDS-PAGE. The results are shown in Table 59. After freeze-thaw, some LMW and HMW were observed, for example in formulation D7 (containing proline).

[0373] Table 59:

[0374]

[0375]

[0376] 4.3. Simulated shear stress study

[0377] The formulation at T0 was subjected to simulated shear stress of 900RPM for 24 hours at room temperature and analyzed for osmotic pressure, pH, A280, RALS, SEC and SDS-PAGE. The results of osmotic pressure, pH, A280 and RALS are shown in Table 60 and Table 61.

[0378] Table 60:

[0379]

[0380] As shown in Table 60, the concentration increased after simulated shear stress, especially for Formulation D2 (containing serine).

[0381] Table 61:

[0382]

[0383] As shown in Table 61, all formulations increased in RALS values ​​after simulated shear stress. Formulations containing alanine and proline (D3 and D7) had the lowest RALS values, indicating improved stability during shear.

[0384] The preparations were subjected to a simulated shear stress of 900 RPM for 24 hours and the total recovery, fusion protein recovery, and the percentage of impurities consisting of HMW and LMW were monitored by SEC-HPLC. The results are shown in Tables 62 and 63. The results show that the preparations containing serine and alanine (D2 and D3) exhibited higher total recovery after simulated shear stress, indicating that the sensitivity to shear stress was relatively small.

[0385] Table 62:

[0386]

[0387] Table 63:

[0388]

[0389] Formulations subjected to simulated shear stress were also analyzed by reducing and non-reducing SDS-PAGE. The results are shown in Table 64. During shear stress, HMW and LMW were observed in all formulations on the gel. Formulations containing arginine and proline (D5 and D7) showed relatively low HMW and LMW percentages during shear stress, indicating improved stability.

[0390] Table 64:

[0391]

[0392] According to the SEC data of thermal stress, the formulations containing arginine and lysine (D5 and D6 containing basic amino acids) showed relatively higher stability compared to other formulations. However, the formulation containing proline (D7) showed a large amount of HMW after freeze-thaw. The formulation containing arginine (D5) showed the largest amount of LMW during simulated shear stress, indicating that it is relatively more sensitive to shear stress. In addition, the results show that the formulations containing alanine and proline (D3 and D7) can resist stirring / simulated shear stress, while the formulations containing arginine and lysine (D5 and D6) can resist thermal stress.

[0393] Example 5 : Stability studies of formulations with different carbohydrate stabilizers, cryoprotectants and / or lyoprotectants

[0394] Formulations of SONE-IL12-ABD were prepared as shown in Table 65 using a SONE-IL12-ABD concentration of approximately 20 μg / ml.

[0395] Table 65:

[0396]

[0397]

[0398] The formulations shown in Table 65 were also lyophilized according to the lyophilization cycle shown in Table 66.

[0399] Table 66:

[0400] step temperature Temperature rise and fall (℃ / min) pressure load environment N / A NA freezing -50℃ 1℃ / min. NA One-time drying -20℃ 1℃ / min 70mTorr Secondary drying +20℃ 1℃ / min 70mTorr

[0401] The liquid formulations were subjected to temperature ramps RALS and IF to obtain the Tagg and Tm of each formulation. The Tm and Tagg values ​​are shown in Table 67. The Tm and Tagg of all formulations were similar. The formulation containing arginine and trehalose (E6) and the formulation containing alanine, arginine and trehalose (E8) had the highest Tm.

[0402] Table 67:

[0403]

[0404] 5.1. Thermal stress study

[0405] Liquid and lyophilized formulations were subjected to heat stress at 40°C for 14 days and monitored for visual appearance, osmotic pressure, pH, A280, RALS, Flowcam, SEC-HPLC, reduced and non-reduced SDS-page, and ELISA. The visual appearance results are shown in Table 68, the osmotic pressure, pH, and RALS results are shown in Table 69 and Table 70, and the A280 results are shown in Table 71 and Table 72.

[0406] As shown in Table 68, all liquid and reconstituted samples were clear, colorless, and free of visible particles. The lyophilized cakes of the formulation containing mannitol (E4) had no cracks or pulling away from the glass. The lyophilized cakes of E-CF and the formulation containing alanine, sucrose, and mannitol (E5) completely collapsed at T14.

[0407] Table 68:

[0408]

[0409] Table 69:

[0410]

[0411]

[0412] Table 70:

[0413]

[0414] Table 71:

[0415]

[0416] Table 72:

[0417]

[0418] As shown in Table 70, RALS was slightly increased in the lyophilized formulation containing alanine and sucrose (E3), the lyophilized formulation containing alanine and mannitol (E4), the lyophilized formulation containing alanine, sucrose and mannitol (E5), and the lyophilized formulation containing arginine and trehalose (E6).

[0419] After 7 and 14 days of incubation at 40°C, the presence of subparticles in the formulations was assessed by Flowcam. As shown in Table 73, after 14 days of incubation, the subvisible particle counts of all liquid formulations increased, among which the particle counts of the formulation containing proline (E7) were relatively high. As shown in Table 74, after 14 days of incubation, the subvisible particle counts of all lyophilized formulations increased, among which the particle counts of the formulation containing proline and trehalose (E7) were relatively high.

[0420] Table 73:

[0421]

[0422] Table 74:

[0423]

[0424] The formulations were heat stressed at 40°C for 14 days and the total recovery, fusion protein recovery, and percentage of impurities consisting of HMW and LMW were monitored by SEC-HPLC. The results are shown in Tables 75 to 80.

[0425] After incubation for 14 days, the %HMW of the liquid preparation containing arginine and trehalose (E6) and the liquid preparation containing alanine, arginine and trehalose (E8) on SEC did not change. The results show that the presence of arginine protects the liquid preparation from the influence of thermal stress. The %LMW of the liquid preparation containing alanine, sucrose and mannitol (E5) is the lowest. The main peak % at T14 of the liquid preparation containing arginine and trehalose (E6) and the liquid preparation containing alanine, arginine and trehalose (E8) is the highest. The results also show that the total recovery of the liquid preparation containing alanine and trehalose (E2) and the liquid preparation containing alanine, arginine and trehalose (E8) is relatively high.

[0426] The lyophilized formulations were relatively more stable after 14 days at 40° C. in terms of total recovery and major peak recovery. E-CF had a slightly higher %HMW, and the formulation containing arginine and trehalose (E6) had relatively lower major peak recovery and %LMW at T14.

[0427] Table 75:

[0428]

[0429]

[0430] Table 76:

[0431]

[0432] Table 77:

[0433]

[0434] Table 78:

[0435]

[0436] Table 79:

[0437]

[0438] Table 80:

[0439]

[0440]

[0441] Liquid and lyophilized formulations E1 (trehalose), E2 (alanine and trehalose), E4 (alanine and mannitol) and E8 (alanine, arginine and trehalose) heat stressed at 40°C for 14 days were also analyzed by reducing and non-reducing SDS-PAGE. Qualitative analysis by silver staining showed that formulation E2 (alanine and trehalose) had the lowest impurities.

[0442] Liquid and lyophilized formulations E1 (trehalose), E2 (alanine and trehalose), E4 (alanine and mannitol) and E8 (alanine, arginine and trehalose) were also analyzed by ELISA. The results are listed in Table 81.

[0443] Table 81:

[0444]

[0445] 5.2. Freeze-thaw studies

[0446] The formulations at T0 were subjected to five freeze-thaw cycles and then analyzed for osmotic pressure, pH, RALS, A280, Flowcam and SEC-HPLC. The results for osmotic pressure, pH, A280 and RALS are shown in Table 81A.

[0447] Table 81A:

[0448]

[0449] As shown in Table 81A, except for formulation E-CF, the concentration decreased slightly after freeze-thaw.

[0450] After five freeze-thaw cycles, the formulations were evaluated by Flowcam for the presence of subparticles. The results are shown in Table 82. Subvisible particles in the 2-10 μm range were increased in all formulations.

[0451] Table 82:

[0452]

[0453] The preparations were subjected to five freeze-thaw cycles and total recovery, fusion protein recovery, and impurity percentages consisting of HMW and LMW were monitored by SEC-HPLC. The results are shown in Tables 83 and 84. The %HMW of each preparation was similar, except for E-CF and the preparation containing trehalose (E1), which had relatively low HMW substances after freeze-thaw. The %LMW content of the preparation containing alanine and sucrose (E3) and the preparation containing alanine, sucrose and mannitol (E5) was the lowest. The preparation E6 containing alanine, arginine and trehalose had the lowest recovery % after freeze-thaw.

[0454] Table 83:

[0455]

[0456] Table 84:

[0457]

[0458] 5.3. Simulated shear stress study

[0459] The formulation at T0 was subjected to simulated shear stress of 900RPM at room temperature for 24 hours and analyzed for osmotic pressure, pH, A280, RALS, SEC and DLS. The results of osmotic pressure, pH, A280 and RALS are shown in Table 85.

[0460] Table 85:

[0461]

[0462] As shown in Table 85, formulation E1 containing trehalose and formulation E2 containing alanine and trehalose have the lowest concentration after simulated shear stress, indicating that there are fewer light scattering particles that affect or interfere with the absorbance measurement at A280. In addition, formulation E2 containing alanine and trehalose has better resistance to shear stress and has the lowest RALS value. Compared with other formulations, formulation E6 containing arginine and trehalose and formulation E8 containing alanine, arginine and trehalose are relatively more sensitive to shear stress, as shown by higher RALS values.

[0463] The preparations were subjected to a simulated shear stress of 900RPM for 24 hours, and the total recovery, fusion protein recovery, and the impurity percentage consisting of HMW and LMW were monitored by SEC-HPLC. The results are shown in Table 86 and Table 87. As monitored by SEC, the %HMW of all liquid preparations increased after shearing. The %LMW of the preparation E1 containing trehalose and the preparation E8 containing alanine, arginine and trehalose increased significantly. The preparation E7 containing proline and trehalose also had the highest main peak %, and the E2 containing alanine and trehalose had the highest total recovery % after shearing, indicating that the sensitivity to shear stress was relatively low.

[0464] Table 86:

[0465]

[0466]

[0467] Table 87:

[0468]

[0469] The formulations subjected to simulated shear stress were also analyzed by DLS. The results are shown in Table 88. Formulations E1 (containing trehalose), E2 (containing alanine and trehalose), E3 (containing alanine and sucrose), and E7 (containing proline and trehalose) had relatively small particle sizes after simulated shear stress, with a diameter of approximately 1.2 nm.

[0470] Table 88:

[0471]

[0472]

[0473] In summary, the results of Example 5 show that arginine can resist heat stress and alanine can resist shear stress. For example, formulation E8 (containing alanine, arginine and trehalose) and formulation E2 (containing alanine and trehalose) stabilize SONE-IL12-ABD, and liquid E2 and E8 formulations have the highest total recovery percentage on SEC after 14 days of incubation.

[0474] Example 6 :Stability study of formulations with different stabilizers

[0475] Formulations of SONE-IL12-ABD were prepared as shown in Table 89 using a SONE-IL12-ABD concentration of approximately 20 μg / ml.

[0476] Table 89:

[0477]

[0478] The formulations shown in Table 89 were also lyophilized according to the lyophilization cycle shown in Table 66.

[0479] The liquid formulations were subjected to temperature ramping RALS and IF to obtain the Tagg and Tm of each formulation. The Tm and Tagg values ​​are shown in Table 90. The Tm and Tagg of all formulations were similar.

[0480] Table 90:

[0481]

[0482]

[0483] 6.1. Thermal stress studies

[0484] Liquid and lyophilized formulations were subjected to heat stress at 40°C for 14 days and monitored for visual appearance, osmotic pressure, pH, A280, RALS, Flowcam, SEC-HPLC, reduced and non-reduced SDS-page, and ELISA. The visual appearance results are shown in Table 91, the osmotic pressure, pH, and RALS results are shown in Table 92 and Table 93, and the A280 results are shown in Table 94 and Table 95.

[0485] As shown in Table 91, all liquids and reconstituted samples were clear, colorless, and free of visible particles. T7 F4 vials contained more trehalose than mannitol and had a defective stopper that prevented lyophilization, but F4's T14 data could be measured. F6 contained more mannitol than trehalose, and the lyophilized cake collapsed completely at T0 and did not change at T14. The cakes of the lyophilized formulations containing alanine and arginine showed improvement compared to the cakes of the lyophilized formulations containing mannitol and trehalose.

[0486] Table 91:

[0487]

[0488] Table 92:

[0489]

[0490]

[0491] Table 93:

[0492]

[0493] *No data.

[0494] Table 94:

[0495]

[0496] Table 95:

[0497]

[0498]

[0499] *No data.

[0500] As shown in Table 93, among the T14 lyophilized samples, the osmotic pressure increased more in F1 (containing more alanine than arginine) and F2 (containing equal amounts of alanine and arginine).

[0501] As shown in Table 94, the concentrations of all liquid formulations were reduced, except for F1 (which contained more alanine than arginine).

[0502] After 7 and 14 days of incubation at 40°C, the formulations were evaluated by Flowcam for the presence of subparticles. As shown in Table 96, after 14 days of incubation, the subvisible particle counts of all liquid formulations increased, with formulation F6 (containing more mannitol than trehalose) showing the largest increase. As shown in Table 97, after 14 days of incubation, the subvisible particle counts of all lyophilized formulations increased.

[0503] Table 96:

[0504]

[0505] Table 97:

[0506]

[0507]

[0508] *No data.

[0509] The formulations were heat stressed at 40°C for 14 days and the total recovery, fusion protein recovery, and percentage of impurities consisting of HMW and LMW were monitored by SEC-HPLC. The results are shown in Tables 98 to 103.

[0510] Liquid formulation F5 containing equal amounts of trehalose and mannitol had the lowest LMW on SEC at T14. Liquid formulations containing arginine had very low HMW at T14. The percentage of the main peak was the highest for formulations containing arginine. The results also showed that formulations containing arginine could resist heat stress.

[0511] The %LMW of all lyophilized formulations decreased on SEC at T14. The %HMW of all lyophilized formulations at T14 was very low. The percentage of the main peak was relatively similar for all lyophilized formulations, with F-LF and F5 (containing equal amounts of trehalose and mannitol) having the highest percentage of the main peak at 96%.

[0512] Table 98:

[0513]

[0514] Table 99:

[0515]

[0516] Table 100:

[0517]

[0518] Table 101:

[0519]

[0520] Table 102: *No data.

[0521] Table 103:

[0522]

[0523] Liquid and lyophilized formulations F-LF, F1, F2, and F3 heat stressed at 40°C for 14 days were also analyzed by reducing and non-reducing SDS-PAGE. Qualitative analysis by silver staining showed that formulation F-LF and the formulation containing mannitol had the lowest impurities.

[0524] Liquid and lyophilized formulations F-LF, F4, F5 and F6 were also analyzed by ELISA. The results are listed in Table 104.

[0525] Table 104:

[0526]

[0527] 6.2. Freeze-thaw studies

[0528] The formulation at T0 was subjected to five freeze-thaw cycles and then analyzed for osmotic pressure, pH, RALS, A280, Flowcam and SEC-HPLC. The results of osmotic pressure, pH, A280 and RALS are shown in Table 105.

[0529] Table 105:

[0530]

[0531] After five freeze-thaw cycles, the formulations were evaluated by Flowcam for the presence of subparticles. The results are shown in Table 106. Subvisible particles in the 2-10 μm range were increased in all formulations.

[0532] Table 106:

[0533]

[0534] The formulations were subjected to five freeze-thaw cycles and monitored by SEC-HPLC for total recovery, fusion protein recovery, and the percentage of impurities consisting of HMW and LMW. The results are shown in Tables 107 and 108. After freeze-thaw, the %LMW of F1 on SEC decreased significantly. The main % after freeze-thaw of all formulations was consistent at around 93%. F-LF containing alanine and trehalose and F4 containing more trehalose than mannitol had the highest percentage of total recovery after freeze-thaw at 90%.

[0535] Table 107:

[0536]

[0537] Table 108:

[0538]

[0539] 6.3. Simulated shear stress study

[0540] At room temperature, the formulation at T0 was subjected to a simulated shear stress of 900RPM for 24 hours and analyzed for osmotic pressure, pH, A280, RALS, SEC and DLS. The results of osmotic pressure, pH, A280 and RALS are shown in Tables 109 and 110.

[0541] Table 109:

[0542]

[0543]

[0544] Table 110:

[0545]

[0546] As shown in Table 109, the formulation concentration increased slightly for all formulations after simulated shear stress. As shown in Table 110, the RALS values ​​for all formulations increased after simulated shear stress. The RALS values ​​for formulations F2 (containing more alanine than arginine) and F3 (containing more arginine than alanine) were very high, indicating that these formulations were sensitive to shear stress.

[0547] The preparations were subjected to a simulated shear stress of 900RPM for 24 hours, and the total recovery, fusion protein recovery, and the impurity percentage consisting of HMW and LMW were monitored by SEC-HPLC. The results are shown in Tables 111 and 112. After the simulated shear stress, the %LMW of F1 containing more alanine than arginine and F3 containing more arginine than alanine on SEC was the lowest. After shearing, the %HMW of all preparations increased, and the main peak % of F-LF containing alanine and trehalose was the highest. After the simulated shear stress, the total recovery % of F-LF containing alanine and trehalose, F4 containing more trehalose than mannitol, and F6 containing more mannitol than trehalose was the highest.

[0548] Table 111:

[0549]

[0550] Table 112:

[0551]

[0552] The formulations subjected to simulated shear stress were also analyzed by DLS. The results are shown in Table 113. The particle sizes of 100% volume on DLS for formulations F-LF containing alanine and trehalose and F4 containing more trehalose than mannitol were approximately 1.2 and 1.5 nm, indicating that these formulations protected SONE-IL12-ABD from shear stress.

[0553] Table 113:

[0554]

[0555]

[0556] In summary, the results of Example 6 demonstrate that arginine can resist thermal stress and alanine can resist shear stress. The results further demonstrate that the formulation containing a combination of alanine and arginine is more stable to thermal stress than to shear stress. In addition, the results demonstrate that the appearance of the lyophilized cake of formulation F-LF is relatively improved compared to the formulation containing a combination of trehalose and mannitol.

[0557] Example 7 :Freeze-dried development

[0558] The SONE-IL12-ABD formulations shown in Table 114 were prepared using a SONE-IL12-ABD concentration of 20 μg / ml.

[0559] Table 114:

[0560]

[0561] The thermal properties of Formulation LF were studied using Modulated Differential Scanning Calorimetry (MDSC). The MDSC conditions and results are shown in Table 115.

[0562] Table 115:

[0563]

[0564] The formulations shown in Table 114 were also lyophilized according to the lyophilization cycles shown in Table 116.

[0565] Table 116:

[0566] step temperature Temperature rise and fall (℃ / min) Keep hours pressure load environment N / A N / A NA freezing -50℃ 1℃ / min. ～4.5 NA One-time drying -30℃ 1℃ / min ～40 70mTorr Secondary drying +20℃ 1℃ / min ～4.0 70mTorr

[0567] The liquid formulation was subjected to temperature rise and fall RALS and IF to obtain the Tagg and Tm of the formulation. The Tm and Tagg values ​​are shown in Table 117.

[0568] Table 117:

[0569]

[0570] 7.1. Thermal stress studies

[0571] Liquid and lyophilized formulations were heat stressed at 40°C for 14 days and monitored for visual appearance, osmotic pressure, pH, A280, RALS, Flowcam, SEC-HPLC, reduced and non-reduced SDS-page, and ELISA. The visual appearance results are shown in Table 118, the osmotic pressure, pH, and RALS results are shown in Tables 119 and 120, and the A280 results are shown in Tables 121 and 122.

[0572] As shown in Table 118, the liquid and reconstituted samples were all clear, colorless, and free of visible particles. The lyophilized cakes of T0, T7, and T14 were all white and slightly out of the glass. Some of the T0 lyophilized cakes had cracks, while others did not.

[0573] Table 118:

[0574]

[0575] Table 119:

[0576]

[0577] Table 120:

[0578]

[0579] Table 121:

[0580]

[0581] Table 122:

[0582]

[0583] After 7 and 14 days of incubation at 40°C, the presence of subparticles in the formulations was assessed by Flowcam. As shown in Table 123, the subvisible particle counts of the liquid formulations increased after 14 days of incubation. As shown in Table 124, the subvisible particle counts of the lyophilized formulations also increased after 14 days of incubation.

[0584] Table 123:

[0585]

[0586] Table 124:

[0587]

[0588] The preparation was subjected to heat stress treatment at 40°C for 14 days, and the total recovery, fusion protein recovery, and the percentage of impurities consisting of HMW and LMW were monitored by SEC-HPLC. The results are shown in Tables 125 to 130. For liquid preparations, at T14, %HMW increased significantly, %LMW decreased, and the main peak % decreased. For lyophilized preparations, at T14, %HMW increased slightly, the main peak % did not change significantly, and %LMW did not change significantly. For lyophilized samples, the recovery was 100% at T14.

[0589] Table 125:

[0590]

[0591] Table 126:

[0592]

[0593]

[0594] Table 127:

[0595]

[0596] Table 128:

[0597]

[0598] Table 129:

[0599]

[0600] Table 130:

[0601]

[0602] Liquid and lyophilized formulations LF subjected to heat stress at 40°C for 14 days were also analyzed by reducing and non-reducing SDS-PAGE. Qualitative analysis by silver staining showed no significant differences between liquid and lyophilized formulations.

[0603] Liquid and lyophilized formulations LF were also analyzed by ELISA. The results are shown in Table 131.

[0604] Table 131:

[0605]

[0606]

[0607] 7.2. Freeze-thaw studies

[0608] Formulation LF at T0 was subjected to five freeze-thaw cycles and then analyzed for osmotic pressure, pH, RALS, A280, Flowcam and SEC-HPLC. The results of osmotic pressure, pH, A280 and RALS are shown in Table 132.

[0609] Table 132:

[0610]

[0611] After five freeze-thaw cycles, the presence of subparticles in Formulation LF was evaluated by Flowcam. The results are shown in Table 133. Subvisible particles increased after freeze-thaw.

[0612] Table 133:

[0613]

[0614] Formulation LF was subjected to five freeze-thaw cycles and the total recovery, fusion protein recovery, and the percentage of impurities consisting of HMW and LMW were monitored by SEC-HPLC. The results are shown in Tables 134 and 135. After freeze-thaw, %LMW was reduced by almost half and %HMW increased slightly.

[0615] Table 134:

[0616]

[0617] Table 135:

[0618]

[0619] 7.3. Simulated shear stress studies

[0620] At room temperature, the formulation LF at T0 was subjected to a simulated shear stress of 900RPM for 24 hours and analyzed for osmotic pressure, pH, A280, RALS, SEC and DLS. The results of osmotic pressure, pH, A280 and RALS are shown in Tables 136 and 137.

[0621] Table 136:

[0622]

[0623] Table 137:

[0624]

[0625] Formulation LF was subjected to a simulated shear stress of 900RPM for 24 hours and the total recovery, fusion protein recovery, and the percentage of impurities consisting of HMW and LMW were monitored by SEC-HPLC. The results are shown in Tables 138 and 139. After the simulated shear stress, the %HMW on SEC increased significantly, the %LMW decreased, and the main peak % decreased.

[0626] Table 138:

[0627]

[0628] Table 139:

[0629]

[0630] Formulation LF subjected to simulated shear stress was also analyzed by DLS. The results are shown in Table 140.

[0631] Table 140:

[0632]

[0633] In summary, the results of Example 7 show that the freeze-dried formulation LF is stable at 40°C for 14 days, and the ELISA test proves that all activity is retained, and there is no increase in HMW or LMW substances.

[0634] Unless otherwise indicated, all numbers used in the specification and claims indicating the amounts of ingredients, properties (e.g., molecular weight), reaction conditions, etc., should be understood as being modified in all cases by the term "about". Therefore, unless otherwise indicated, the numerical parameters listed in the specification and the appended claims are approximate and can vary depending on the desired properties to be obtained by the present disclosure. At least, without attempting to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be interpreted in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0635] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the present disclosure are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0636] The terms "a", "an", "the" and similar references used in the context of describing the present disclosure (especially in the context of the following claims) should be interpreted as covering the singular and the plural, unless otherwise stated herein or the context is clearly contradictory. The recitation of numerical ranges herein is intended only as a shorthand method of individually referring to each individual value within the range. Unless otherwise stated herein, each individual value is incorporated into the specification as if it were individually stated herein. All methods described herein can be performed in any suitable order, unless otherwise stated herein or the context is clearly contradictory. The use of any and all examples or exemplary language (e.g., "for example") provided herein is intended only to better illustrate the present disclosure and does not limit the scope of the claimed disclosure. No language in the specification should be interpreted as indicating any non-required element essential to the practice of the present disclosure.

[0637] The grouping of alternative elements or embodiments of the present disclosure disclosed herein should not be considered as limiting. Each group member can be quoted and claimed separately, or can be quoted and claimed in any combination with other members or other elements in the group present in this article. It is foreseeable that one or more members in the group can be included in or deleted from the group for convenience and / or patentability reasons. When any such inclusion or deletion occurs, the specification is deemed to contain the modified group, thereby satisfying the written description of all Markush groups used in the appended claims.

[0638] Certain embodiments of the present disclosure are described herein, including the best mode of implementing the present disclosure known to the inventor. Of course, those of ordinary skill in the art will clearly understand the variations of these described embodiments after reading the above description. The inventors hope that skilled technicians can adopt such variations as appropriate, and the inventors hope that the present disclosure will be implemented in a manner different from that specifically described herein. Therefore, the present disclosure includes all modifications and equivalents of the subject matter described in the appended claims as allowed by applicable law. In addition, unless otherwise stated herein or the context is clearly contradictory, the present disclosure covers any combination of the above elements in all possible variations thereof.

[0639] Specific embodiments disclosed herein may be further limited in the claims using language such as "consisting of" or "consisting essentially of. When used in a claim, whether as filed or added by amendment, the transition term "consisting of" excludes any element, step, or ingredient not specified in the claim. The transition term "consisting essentially of" limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristics. The claimed embodiments of the present disclosure are inherently or expressly described and enabled herein.

[0640] It should be understood that the embodiments of the present disclosure disclosed herein are only used to illustrate the principles of the present disclosure. Other modifications that can be adopted are within the scope of the present disclosure. Therefore, as an example, but not as a limitation, alternative configurations of the present disclosure can be utilized according to the teachings of this article. Therefore, the present disclosure is not limited to the specific contents shown and described.

[0641] Although the present disclosure has been described and illustrated herein by reference to various specific materials, procedures and examples, it should be understood that the present disclosure is not limited to the specific combination of materials and procedures selected for this purpose. It will be appreciated by those skilled in the art that a variety of changes in such details may be implied. This specification and examples are intended to be considered as exemplary only, and the true scope and spirit of the present disclosure are indicated by the following claims. All references, patents and patent applications cited in this application are incorporated herein by reference in their entirety.

Claims

1. A stable liquid pharmaceutical preparation comprising: - about 10 μg / mL to about 50 μg / mL of a fusion protein of SEQ ID NO: 11; - about 25 mM to about 100 mM alanine; - about 100 mM to about 400 mM trehalose; - from about 10 mM to about 50 mM glycylglycine; - from about 0.01% to about 0.04% polysorbate 20 (v / v); and - about 5 μM to about 20 μM diethylenetriaminepentaacetic acid (DTPA), wherein the pH of the formulation is from about 6.8 to about 8.

0.

2. The liquid formulation of claim 1, wherein the formulation comprises about 10 μg / mL to about 30 μg / mL, about 15 μg / mL to about 25 μg / mL, about 10 μg / mL to about 15 μg / mL, about 15 μg / mL to about 20 μg / mL, about 20 μg / mL to about 25 μg / mL, about 25 μg / mL to about 30 μg / mL, about 30 μg / mL to about 35 μg / mL, about 35 μg / mL to about 40 μg / mL, about 40 μg / mL to about 45 μg / mL, about 45 μg / mL to about 50 μg / mL, about 10 μg / mL, about 15 μg / mL, about 20 μg / mL, about 25 μg / mL, about 30 μg / mL, about 35 μg / mL, about 40 μg / mL, about 45 μg / mL and / or about 50 μg / mL of SEQ ID The fusion protein of SEQ ID NO: 11, preferably wherein the preparation comprises the fusion protein of SEQ ID NO: 11 in an amount of about 10 μg / mL to about 30 μg / mL. 3 . The liquid formulation of claim 1 , wherein the formulation comprises about 20 μg / mL of the fusion protein of SEQ ID NO:

11.

4. The liquid formulation of any one of claims 1 to 3, wherein the formulation comprises about 25 mM to about 75 mM alanine, about 40 mM to about 60 mM alanine, about 25 mM to about 30 mM alanine, about 30 mM to about 40 mM alanine, about 40 mM to about 50 mM alanine, about 50 mM to about 60 mM alanine, about 60 mM to about 70 mM alanine, about 70 mM to about 80 mM alanine, about 80 mM to about 90 mM alanine, about 90 mM to about 100 mM alanine, about 25 mM alanine, about 30 mM alanine, about 40 mM alanine, about 50 mM alanine, about 60 mM alanine, about 70 mM alanine, about 80 mM alanine, about 90 mM alanine and / or about 100 mM alanine, preferably wherein the formulation comprises about 50 mM alanine.

5. The liquid formulation according to any one of claims 1 to 4, wherein the formulation comprises about 100 mM to about 300 mM trehalose, about 150 mM to about 250 mM trehalose, about 100 mM to about 150 mM trehalose, about 150 mM to about 200 mM trehalose, about 200 mM to about 250 mM trehalose, about 250 mM to about 300 mM trehalose, about 300 mM to about 350 mM trehalose, about 350 mM to about 400 mM trehalose, about 100 mM trehalose, about 150 mM trehalose, about 200 mM trehalose, about 250 mM trehalose, about 300 mM trehalose, about 350 mM trehalose and / or about 400 mM trehalose, preferably wherein the formulation comprises about 200 mM trehalose.

6. The liquid formulation of any one of claims 1 to 5, wherein the formulation comprises about 10 mM to about 40 mM glycylglycine, about 10 mM to about 30 mM glycylglycine, about 10 mM to about 15 mM glycylglycine, about 15 mM to about 20 mM glycylglycine, about 20 mM to about 25 mM glycylglycine, about 25 mM to about 30 mM glycylglycine, about 35 mM to about 40 mM glycylglycine, about 40 mM to about About 45 mM glycylglycine, about 45 mM to about 50 mM glycylglycine, about 10 mM glycylglycine, about 15 mM glycylglycine, about 20 mM glycylglycine, about 25 mM glycylglycine, about 30 mM glycylglycine, about 35 mM glycylglycine, about 40 mM glycylglycine, about 45 mM glycylglycine and / or about 50 mM glycylglycine, preferably wherein the formulation comprises about 25 mM glycylglycine.

7. The liquid formulation according to any one of claims 1 to 6, wherein the formulation comprises about 0.01% to about 0.03% polysorbate 20 (v / v), about 0.01% to about 0.02% polysorbate 20 (v / v), about 0.02% to about 0.03% polysorbate 20 (v / v), about 0.03% to about 0.04% polysorbate 20 (v / v), about 0.01% polysorbate 20 (v / v), about 0.02% polysorbate 20 (v / v), about 0.03% polysorbate 20 (v / v) and / or about 0.04% polysorbate 20 (v / v), preferably wherein the formulation comprises about 0.02% polysorbate 20 (v / v).

8. The liquid formulation of any one of claims 1 to 7, wherein the formulation comprises about 5 μM to about 15 μM DTPA, about 5 μM to about 10 μM DTPA, about 10 μM to about 15 μM DTPA, about 15 μM to about 20 μM DTPA, about 5 μM DTPA, about 10 μM DTPA, about 15 μM DTPA and / or about 20 μM DTPA, preferably wherein the formulation comprises about 10 μM DTPA.

9. The liquid formulation of any one of claims 1 to 8, wherein the formulation has a pH of about 7.0 to about 7.8, about 7.2 to about 7.8, about 7.4 to about 7.6, about 7.0 to about 7.2, about 7.2 to about 7.4, about 7.4 to about 7.6, about 7.6 to about 7.8, about 7.8 to about 8.0, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9 and / or about 8.0, preferably wherein the formulation has a pH of about 7.

5.

10. A liquid pharmaceutical preparation comprising: - about 20 μg / mL of the fusion protein of SEQ ID NO: 11; - about 50 mM alanine; - about 200 mM trehalose; - about 25 mM glycylglycine; - about 0.02% polysorbate 20 (v / v); and - about 10 μM DTPA, wherein the formulation has a pH of about 7.

5.

11. The liquid preparation according to any one of claims 1 to 10, wherein the preparation is a reconstituted preparation. The liquid preparation according to claim 11 , wherein the reconstituted preparation is reconstituted from a lyophilized preparation.

13. A solid pharmaceutical preparation comprising: - about 0.01% to about 0.03% (w / w) of a fusion protein of SEQ ID NO: 11; - about 2% to about 8% alanine (w / w); - about 50% to about 95% trehalose (w / w); - about 2% to about 8% glycylglycine (w / w); - from about 0.1% to about 0.4% polysorbate 20 (w / w); and - from about 0.002% to about 0.006% diethylenetriaminepentaacetic acid (DTPA) (w / w).

14. The solid pharmaceutical preparation according to claim 13, comprising: - about 0.024 to about 0.028% (w / w) of a fusion protein of SEQ ID NO: 11; - about 4.0% to about 5.0% alanine (w / w); - about 70% to about 80% trehalose (w / w); - about 3.0% to about 4.0% glycylglycine (w / w); - from about 0.15% to about 0.30% polysorbate 20 (w / w); and - about 0.0045% to about 0.0055% DTPA (w / w).

15. The preparation according to claim 13 or 14, wherein the preparation is a lyophilized preparation.

16. The formulation according to any one of the preceding claims, wherein the formulation is suitable for parenteral administration.

17. The formulation according to any one of claims 1 to 15, wherein the formulation is suitable for intravenous administration.

18. The formulation according to any one of claims 1 to 15, wherein the formulation is suitable for subcutaneous administration.

19. A method of treating an IL-12-related disease or disorder in a subject in need thereof, the method comprising administering to the subject the formulation of any one of claims 1 to 15.

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