IL-12-Albumin Binding Domain Fusion Protein Formulation and Method of Use Thereof

Abstract

The present disclosure provides a formulation of an IL-12-albumin binding domain (ABD) fusion protein comprising a fusion protein of about 10 μg / mL to about 50 μg / mL, 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), and the formulation has a pH of about 6.8 to about 8.0. Such a formulation may be useful for treating and / or preventing an IL-12 related disease or disorder in a subject in need of treatment of an IL-12 related disease or disorder.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 346,368, filed on May 27, 2023, the entire contents of which are incorporated herein by reference.

[0002] The present disclosure generally provides IL - 12 - albumin binding domain (ABD) fusion protein formulations and methods of using them, including for the treatment of IL - 12 - related diseases and / or disorders.

Background Art

[0003] IL - 12 can mediate immune effector functions in a manner compatible with enhancing pro - inflammatory endogenous antitumor immune responses. (See, for example, Boggio et al., J Exp Med 188:589 - 96 (1998), Cavallo et al., Cancer Res 59:414 - 21 (1999), Yu et al., Int Immunol 8:855 - 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 T - cell secretion of IFNγ mediated by IL - 12 can reverse T - cell anergy and confer effector T - cell resistance against immunosuppressive regulatory T cells. The ability of IL - 12 not only to activate the adaptive and innate immune systems but also to further regulate other immune - hostile tumor microenvironments makes IL - 12 an ideal candidate for tumor immunotherapy.

[0004] Serum albumin has a long half-life in the range of 2 to 4 weeks due to recycling via the neonatal Fc receptor (FcRn). Albumin is taken up by endothelial cells via macropinocytosis and binds to FcRn in a pH-dependent manner in the acidic environment of early endosomes. Albumin-FcRn binding avoids albumin molecules from degradation within the lysosomal compartment, redirects albumin molecules to the plasma membrane, where they are released into the plasma due to neutral pH.

[0005] The albumin-binding domain (ABD) fusion proteins described in U.S. Patent No. 11,028,166 are useful for extending the half-life of biopharmaceuticals (e.g., interleukins and antibodies). Such ABDs do not compete with FcRN for albumin binding and bind to albumin in a pH range that allows the ABD to also undergo FcRn-driven endosomal albumin recycling when bound to albumin. Thus, biopharmaceuticals containing ABD can avoid lysosomal degradation using the albumin-FcRn pathway and as a result exhibit a longer serum half-life than their counterparts lacking ABD. Furthermore, such ABD-containing therapeutics preferentially localize to tumors known to contain high levels of serum albumin. Thus, such ABD-containing therapeutics are particularly useful for the treatment of cancer.

[0006] The therapeutic use of ABD fusion proteins is facilitated by formulations that maintain the stability of the fusion proteins under various conditions. It is important for therapeutic formulations to allow storage without unacceptable loss of activity of the active protein, minimize the accumulation of unwanted products such as aggregates or degradation species (e.g., fragmented, oxidized, deamidated, or isomerized species), accept the appropriate concentration of the protein, and contain no components that are incompatible with therapeutic use. Therefore, there is a need in the art for stable pharmaceutical formulations containing IL-12-ABD fusion proteins suitable for therapeutic use. SUMMARY OF THE INVENTION

[0007] The present disclosure provides an IL12-ABD fusion protein formulation that is stable like no other in both liquid and solid (e.g., lyophilized) forms at low concentrations of the fusion protein. Such a stable liquid pharmaceutical formulation comprises the IL12-ABD fusion protein of SEQ ID NO: 11 at about 10 μg / mL to about 50 μg / mL, alanine at about 25 mM to about 100 mM, trehalose at about 100 mM to about 400 mM, glycylglycine at about 10 mM to about 50 mM, polysorbate 20 at about 0.01% to about 0.04% (v / v), and diethylenetriaminepentaacetic acid (DTPA) at about 5 μM to about 20 μM, and the formulation has a pH of about 6.8 to about 8.0.

[0008] 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, 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.

[0009] 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.

[0010] In some embodiments, the liquid formulation comprises the fusion protein of SEQ ID NO: 11 at about 20 μg / mL.

[0011] In some embodiments, the liquid formulation comprises from about 25 mM to about 75 mM alanine, from about 40 mM to about 60 mM alanine, from about 25 mM to about 30 mM alanine, from about 30 mM to about 40 mM alanine, from about 40 mM to about 50 mM alanine, from about 50 mM to about 60 mM alanine, from about 60 mM to about 70 mM alanine, from about 70 mM to about 80 mM alanine, from about 80 mM to about 90 mM alanine, from 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.

[0012] In some embodiments, the liquid formulation comprises about 50 mM alanine.

[0013] In some embodiments, the liquid formulation comprises from about 100 mM to about 300 mM trehalose, from about 150 mM to about 250 mM trehalose, from about 100 mM to about 150 mM trehalose, from about 150 mM to about 200 mM trehalose, from about 200 mM to about 250 mM trehalose, from about 250 mM to about 300 mM trehalose, from about 300 mM to about 350 mM trehalose, from 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.

[0014] In some embodiments, the liquid formulation comprises about 200 mM trehalose.

[0015] In some embodiments, the liquid formulation comprises from about 10 mM to about 40 mM of glycylglycine, from about 10 mM to about 30 mM of glycylglycine, from about 10 mM to about 15 mM of glycylglycine, from about 15 mM to about 20 mM of glycylglycine, from about 20 mM to about 25 mM of glycylglycine, from about 25 mM to about 30 mM of glycylglycine, from about 35 mM to about 40 mM of glycylglycine, from about 40 mM to about 45 mM of glycylglycine, from about 45 mM to about 50 mM of glycylglycine, 10 mM of glycylglycine, 15 mM of glycylglycine, 20 mM of glycylglycine, 25 mM of glycylglycine, 30 mM of glycylglycine, 35 mM of glycylglycine, 40 mM of glycylglycine, 45 mM of glycylglycine, and / or 50 mM of glycylglycine.

[0016] In some embodiments, the liquid formulation comprises 25 mM of glycylglycine.

[0017] In some embodiments, the liquid formulation comprises from about 0.01% to about 0.03% of polysorbate 20 (v / v), from about 0.01% to about 0.02% of polysorbate 20 (v / v), from about 0.02% to about 0.03% of polysorbate 20 (v / v), from about 0.03% to about 0.04% of polysorbate 20 (v / v), 0.01% of polysorbate 20 (v / v), 0.02% of polysorbate 20 (v / v), 0.03% of polysorbate 20 (v / v), and / or 0.04% of polysorbate 20 (v / v).

[0018] In some embodiments, the liquid formulation comprises 0.02% of polysorbate 20 (v / v).

[0019] In some embodiments, the liquid formulation comprises from about 5 μM to about 15 μM of DTPA, from about 5 μM to about 10 μM of DTPA, from about 10 μM to about 15 μM of DTPA, from about 15 μM to about 20 μM of DTPA, 5 μM of DTPA, 10 μM of DTPA, 15 μM of DTPA, and / or 20 μM of DTPA.

[0020] In some embodiments, the liquid formulation comprises about 10 μM of DTPA.

[0021] 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.

[0022] In some embodiments, the liquid formulation has a pH of about 7.5.

[0023] In some embodiments, the liquid pharmaceutical formulation comprises about 20 μg / mL of the fusion protein of SEQ ID NO: 11, about 50 mM of alanine, about 200 mM of trehalose, about 25 mM of glycylglycine, about 0.02% of polysorbate 20 (v / v), and about 10 μM of DTPA, and the formulation has a pH of about 7.5.

[0024] In some embodiments, the liquid formulation is a reconstituted formulation.

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

[0026] 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% of alanine (w / w), about 50% to about 95% of trehalose (w / w), about 2% to about 8% of glycylglycine (w / w), about 0.1% to about 0.4% of polysorbate 20 (w / w), and about 0.002% to about 0.006% of diethylenetriaminepentaacetic acid (DTPA) (w / w).

[0027] In some embodiments, the solid pharmaceutical preparation comprises from about 0.024% to about 0.028% (w / w) of the fusion protein of SEQ ID NO: 11, from about 4.0% and about 5.0% of alanine (w / w), from about 70% to about 80% of trehalose (w / w), from about 3.0% to about 4.0% of glycylglycine (w / w), from about 0.15% to about 0.30% of polysorbate 20 (w / w), and from about 0.0045% to about 0.0055% of DTPA (w / w).

[0028] In some embodiments, the solid preparation is a lyophilized preparation.

[0029] In some embodiments, the preparation is suitable for parenteral administration.

[0030] In some embodiments, the preparation is suitable for intravenous administration.

[0031] In some embodiments, the preparation is suitable for subcutaneous administration.

[0032] 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 preparation disclosed herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0033]

Figure 1

Figure 2

[0034] 1.1 Definitions The following definitions are included to provide a clear and consistent understanding of the specification and claims. As used herein, the recited terms have the following meanings. All other terms and phrases used herein have their ordinary meanings as would be understood by one of ordinary skill in the art. Such ordinary meanings can be obtained by reference to technical dictionaries such as Hawley’s Condensed Chemical Dictionary 14th Edition, by R.J. Lewis, John Wiley & Sons, New York, N.Y., 2001.

[0035] References to “one embodiment,” “an embodiment,” etc. in this specification indicate that the described embodiment may include a particular aspect, feature, structure, part, or characteristic, but not that all embodiments necessarily include that aspect, feature, structure, part, or characteristic. Further, such phrases may refer to the same embodiment mentioned elsewhere in this specification, but not always. Further, when a particular aspect, feature, structure, part, or characteristic is described in connection with an embodiment, whether or not explicitly described, it is within the knowledge of one of ordinary skill in the art to affect or associate such aspect, feature, structure, part, or characteristic with other embodiments.

[0036] Unless otherwise specified in the context, the singular forms "a", "an", and "the" include plural referents. Thus, for example, a reference to "a formulation" includes a plurality of such formulations such that formulation X includes a plurality of formulation Xs. It should be further noted that the claims may be drafted to exclude any optional elements. Thus, this description is intended to serve as a criterion prior to the use of exclusive terms such as "merely", "only", etc. in connection with any element described herein and / or the use of an enumeration of claim elements or "negative" limitations.

[0037] The term "and / or" means any one of the items to which this term is associated, any combination of the items, or all of the items. The phrases "one or more" and "at least one" are readily understood by those skilled in the art, especially when read in the context of their use. For example, the phrase can mean 1, 2, 3, 4, 5, 6, 10, 100, or any upper limit.

[0038] A "stable" IL12-ABD fusion protein formulation is one in which the IL12-ABD fusion protein substantially retains its physical and / or chemical stability and / or its biological activity upon storage. The storage period is generally selected based on the intended shelf life of the formulation. Various analytical techniques for measuring the stability of proteins are available in the art. Examples of analytical techniques are described below. "Substantially retains" is intended to mean a retention of 85% or more, such as at least 90% retention or at least 95% retention.

[0039] The IL12-ABD fusion protein "retains its physical stability" in a pharmaceutical formulation if it shows no significant physical changes, such as aggregation, precipitation, and / or denaturation, when measured by visual inspection of color and / or transparency, or by UV light scattering, or by size exclusion chromatography.

[0040] If no significant chemical changes in the protein are shown, the IL12-ABD fusion protein "retains its chemical stability" in the pharmaceutical formulation. Chemical stability can be evaluated by detecting and quantifying the chemically modified forms of the protein. Chemical modifications can involve size changes (e.g., clipping) that can be evaluated using, for example, size exclusion chromatography, SDS-PAGE, and / or matrix-assisted laser desorption ionization / time-of-flight mass spectrometry (MALDI / TOF MS). Other types of chemical modifications include, for example, charge modifications (e.g., resulting from deamidation, oxidation, and / or isomerization) that can be evaluated by ion exchange chromatography.

[0041] If the biological activity of the protein at a given time has not changed significantly from the biological activity shown at the time the pharmaceutical formulation was prepared, the IL12-ABD fusion protein "retains its biological activity" in the pharmaceutical formulation. The "biological activity" of the IL12-ABD fusion protein refers to the ability of the protein to produce a measurable biological response that can be measured in vitro or in vivo.

[0042] "Pharmaceutical formulation" refers to a preparation that does not contain an amount of a component that is toxic to an unacceptable degree to the patient to whom the formulation is administered.

[0043] The "percent 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 aligning the sequences to achieve the maximum sequence identity percentage and introducing gaps as necessary. Alignment for the purpose of determining the percent identity of amino acid sequences can be achieved in various ways within the art using publicly available computer software such as, for example, BLAST, BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, CLUSTAL OMEGA, or MUSCLE software. One of ordinary skill in the art can determine appropriate parameters for aligning the sequences, including any algorithms necessary to achieve the maximum alignment over the full length of the sequences being compared. Unless otherwise specified, the percent sequence identity is determined using the BLAST algorithm with default parameters.

[0044] "Subject" means a human or non-human mammal including, but not limited to, cows, horses, dogs, sheep, cats, and rodents including mouse and rat species. "Patient" is a human subject.

[0045] As used herein, the terms "treat", "treating", "treatment", etc. refer to reducing or alleviating a disorder and / or associated symptoms or signs, or slowing or stopping its progression. It will be understood that treating a disorder or condition does not necessarily require complete elimination of the associated disorder, symptom, or sign, although this is not excluded.

[0046] As used in this disclosure, the terms "compriss", "comprising", "containing", "having", "includes", "including", and their linguistic variations have the meaning ascribed to them in United States patent law and, in addition to their explicit recitation, allow for the presence of additional components.

[0047] As will be understood by those skilled in the art, all numbers, including those representing amounts of ingredients, properties such as molecular weights, and reaction conditions, etc., are approximate values and are to be understood as optionally modified by the term "about" in all cases. These values can vary depending on the desired properties required to be obtained by those skilled in the art using the teachings of the description herein. Also, it is understood that such values inherently include variations that necessarily result from the standard deviations found in each test measurement. When a value is represented as an approximate value, it will be understood that the use of the antecedent "about" also forms a further aspect with a specific value without the modifying term "about".

[0048] The terms "about" and "approximately" are used interchangeably. Both terms can refer to a variation of ±1%, ±5%, or ±10% of the specified value. For example, "about 50" percent can, in some embodiments, have a variation of 45 - 55 percent, or otherwise be as defined by a particular claim. With respect to integer ranges, the term "about" can include one or two integers greater than and / or less than the recited integer at each end of the range. Unless otherwise indicated herein, the terms "about" and "approximately" are intended to include values proximate to the recited ranges that are equivalent with respect to the functionality of the individual ingredients, compositions, or embodiments, e.g., weight percentages. The terms "about" and "approximately" can also modify the endpoints of the recited ranges as discussed above in this paragraph.

[0049] As will be understood by those skilled in the art, for all purposes, and particularly in providing a written description, all ranges recited herein also include any and all possible subranges and combinations thereof, as well as the individual values that make up the range, particularly integer values. Thus, it is understood that each unit between two particular units is also disclosed. For example, if 10 to 15 is disclosed, then 11, 12, 13, and 14 are also disclosed, individually and as part of the range. Any recited range (e.g., weight percentage or carbon group) includes each specific value, integer, fraction, or identity within that range. Any recited range can be readily recognized as sufficiently describing and enabling the same range to be divided into at least equal halves, thirds, quarters, fifths, or tenths. By way of non-limiting example, each range discussed herein can be readily divided into lower thirds, middle thirds, and upper thirds, etc. Also, as will be understood by those skilled in the art, all language such as "maximum", "at least", "greater than", "less than", "more", "or more than" includes the recited numbers, and such terms refer to ranges that can later be divided into subranges as discussed above. In the same manner, all ratios recited herein also include all subratios that fall within the broader ratio. Thus, the specific values recited for radicals, substituents, and ranges are for illustrative purposes only and do not exclude other defined values or other values within the ranges defined for radicals, substituents, and ranges. It will be further understood that each endpoint of a range is significant both in relation to the other endpoint and independently of the other endpoint.

[0050] The present disclosure provides ranges, limits, and deviations for variables such as volume, mass, percentage, ratio, etc. Those skilled in the art will understand that, for example, a range from "number 1" to "number 2" means a continuous range of numbers including integers and decimals. 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 1.01, 1.02, 1.03, etc. When the disclosed variable is a number less than "number 10", it means a continuous range including integers and decimals less than number 10, as discussed above.

[0051] Similarly, when the disclosed variable is a number greater than "number 10", it means a continuous range including integers and decimals greater than number 10.

[0052] These ranges can be modified by the term "about", the meaning of which has been explained above.

[0053] Those skilled in the art will also readily recognize that when members are grouped together in a common manner such as a Markush group, the present invention encompasses not only the entire listed group as a whole, but also each member of the group individually and all possible subgroups of the main group. In addition, for all purposes, the present invention encompasses not only the main group, but also the main group in which one or more of the group members are absent. Thus, the present invention contemplates an express exclusion of any one or more of the members of the listed group. Accordingly, a proviso may apply to any of the disclosed categories or embodiments, thereby excluding any one or more of the listed elements, species, or embodiments from such category or embodiment, for example, for use in an express negative limitation.

[0054] 1.2. IL12-ABD fusion protein Interleukin 12 (also known as IL-12 or IL12) refers to an interleukin that is a heterodimeric cytokine encoded by the IL-12A and IL-12B genes (Genbank accession numbers: NM_000882 (IL-12A) and NM_002187 (IL-12B)). IL-12 is composed of a bundle of four alpha helices and is involved in the differentiation of naive 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 the production of interferon gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) from T cells and natural killer (NK) cells and can reduce the IL-4-mediated suppression of IFN-γ. IL-12 can further mediate the enhancement of the cytotoxic activity of NK cells and CD8+ cytotoxic T lymphocytes. Furthermore, IL-12 may 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. Although not bound by any particular theory of action, it is thought that IL-12 can be used to treat cancer through its ability to induce an immune response and its anti-angiogenic activity.

[0055] In certain embodiments, IL-12 is a single-chain IL-12 polypeptide comprising an IL-12 p35 subunit attached to an IL-12 p40 subunit. Such single-chain polypeptides of IL-12 advantageously retain one or more of the biological activities of wild-type IL-12. In some embodiments, the single-chain IL-12 polypeptides described herein follow the formula (p40)-(L)-(p35) from the N-terminus to the C-terminus, where "p40" is the IL-12 p40 subunit, "p35" is the IL-12 p35 subunit, and L is a linker. In other embodiments, the single-chain IL-12 follows the formula (p35)-(L)-(p40) from the N-terminus to the C-terminus. Any suitable linker can be used in the single-chain IL-12 polypeptides, including those described herein. Suitable linkers include, for example, a linker having the amino acid sequence (GGGGS) x where x is an integer from 1 to 10. Other suitable linkers include, for example, the amino acid sequence GGGGGGS. Exemplary linkers for single-chain IL-12 that can be used with the subject single-chain IL-12 polypeptides are also described in Lieschke et al., Nature Biotechnology 15:35-40 (1997), which is hereby incorporated by reference in its entirety, particularly for the teachings of IL-12 polypeptide linkers.

[0056] The IL12-ABD fusion proteins provided herein comprise an albumin-binding domain attached to an IL12 fusion partner via a linker. As discussed herein, the subject ABD fusion proteins are capable of undergoing FcRn-mediated endosomal recycling and thus advantageously exhibit an extended half-life compared to counterparts that do not contain such ABDs. The amino acid sequences of ABDs that include vhCDR1, vhCDR2, vhCDR3, vlCDR1, vlCDR2, vlCDR3, variable heavy chain, and variable light chain sequences are disclosed, for example, in FIG. 1.

[0057] 1.3. Formulations In one aspect, provided herein are aqueous liquid or solid formulations (pharmaceutical compositions), such as lyophilized formulations, comprising an IL12-ABD fusion protein. In certain embodiments, the formulation is a reconstituted formulation reconstituted from a lyophilized formulation. In certain embodiments, the formulation is sterile. In certain embodiments, the formulation is stable. In certain embodiments, the formulation further comprises at least one free amino acid. In certain embodiments, the formulation further comprises at least one surfactant. In certain embodiments, the formulation further comprises at least one buffering component. In certain embodiments, the formulation further comprises at least one chelating agent. In certain embodiments, the formulation comprises at least one sugar. In certain embodiments, the formulation comprises an IL12-ABD fusion protein. In certain embodiments, the formulation comprises at least one free amino acid, at least one surfactant, at least one buffering component, at least one chelating agent, at least one sugar, and an IL12-ABD fusion protein.

[0058] In certain embodiments, the liquid formulation comprises from about 10 μg / mL to about 50 μg / mL of an IL12-ABD fusion protein, from about 25 mM to about 100 mM of alanine, from about 100 mM to about 400 mM of trehalose, from about 10 mM to about 50 mM of glycylglycine, from about 0.01% to about 0.04% of polysorbate 20 (v / v), and from about 5 μM to about 20 μM of diethylenetriaminepentaacetic acid (DTPA), and the formulation has a pH of from about 6.8 to about 8.0. In certain embodiments, the liquid formulation comprises from about 20 μg / mL to about 50 μg / mL of an IL12-ABD fusion protein, about 50 mM of alanine, about 200 mM of trehalose, about 25 mM of glycylglycine, about 0.02% of polysorbate 20 (v / v), and about 10 μM of DTPA, and the formulation has a pH of about 7.5. In certain embodiments, the liquid formulation comprises about 20 μg / mL of an IL12-ABD fusion protein, about 50 mM of alanine, about 200 mM of trehalose, about 25 mM of glycylglycine, about 0.02% of polysorbate 20 (v / v), and about 10 μM of DTPA, and the formulation has a pH of about 7.5.

[0059] In certain embodiments, the solid formulation comprises from about 0.01% to about 0.03% (w / w) fusion protein, from about 2% to about 8% alanine (w / w), from about 50% to about 95% trehalose (w / w), from 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). In certain embodiments, the solid formulation comprises from about 0.024 to about 0.028% (w / w) fusion protein, from about 4.0% and about 5.0% alanine (w / w), from about 70% to about 80% trehalose (w / w), from about 3.0% to about 4.0% glycylglycine (w / w), from about 0.15% to about 0.30% polysorbate 20 (w / w), and from about 0.0045% to about 0.0055% DTPA (w / w). In certain embodiments, the weight percentages are w / w of the lyophilized formulation.

[0060] 1.3.1. Concentration of IL12-ABD fusion protein In certain embodiments, the concentration of IL12-ABD in the liquid formulation is from about 10 μg / mL to about 50 μg / mL, from about 10 μg / mL to about 30 μg / mL, from about 15 μg / mL to about 25 μg / mL, from about 10 μg / mL to about 15 μg / mL, from about 15 μg / mL to about 20 μg / mL, from about 20 μg / mL to about 25 μg / mL, from about 25 μg / mL to about 30 μg / mL, from about 30 μg / mL to about 35 μg / mL, from about 35 μg / mL to about 40 μg / mL, from about 40 μg / mL to about 45 μg / mL, from 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.

[0061] In certain embodiments, the concentration of IL12-ABD in the liquid formulation is from about 10 μg / mL to about 50 μg / mL. In certain embodiments, the concentration of IL12-ABD in the liquid formulation is from about 10 μg / mL to about 30 μg / mL. In certain embodiments, the concentration of IL12-ABD in the liquid formulation is about 20 μg / mL.

[0062] In certain embodiments, the IL12-ABD concentration in the solid formulation is from about 0.01% to about 0.03% (w / w), from about 0.015% to about 0.03% (w / w), from about 0.02% to about 0.03% (w / w), from about 0.024% to about 0.028% (w / w), from about 0.01% to about 0.015% (w / w), from about 0.015% to about 0.02% (w / w), from about 0.02% to about 0.022% (w / w), from about 0.022% to about 0.024% (w / w), from about 0.024% to about 0.026% (w / w), from about 0.026% to about 0.028% (w / w), from 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).

[0063] In certain embodiments, the IL12-ABD concentration in the solid formulation is from about 0.01% to about 0.03% (w / w). In certain embodiments, the IL12-ABD concentration in the solid formulation is from about 0.024% to about 0.028% (w / w). In certain embodiments, the IL12-ABD concentration in the solid formulation is about 0.026% (w / w).

[0064] In certain embodiments, the amount of IL12-ABD in the liquid or solid formulation is at least 10 μg, such as 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 certain embodiments, the amount of IL12-ABD in the liquid or solid formulation is about 10 μg to about 200 μg, about 20 μg to about 180 μg, about 30 μg to about 160 μg, about 40 μg to about 150 μg, about 50 μg to about 140 μg, about 60 μg to about 130 μg, about 80 μg to about 120 μg, about 90 μg to about 110 μg, about 10 μg to about 20 μg, about 20 μg to about 30 μg, about 30 μg to about 40 μg, about 40 μg to about 50 μg, about 50 μg to about 60 μg, about 60 μg to about 70 μg, about 70 μg to about 80 μg, about 80 μg to about 90 μg, about 90 μg to about 100 μg, about 100 μg to about 110 μg, about 110 μg to about 120 μg, about 120 μg to about 130 μg, about 130 μg to about 140 μg, about 140 μg to about 150 μg, about 150 μg to about 160 μg, about 160 μg to about 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.

[0065] 1.3.2 Chelating agent In certain embodiments, the formulation comprises a chelating agent.

[0066] In certain embodiments, the chelating agent in the liquid formulation is at a concentration of 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.

[0067] In certain embodiments, the chelating agent in the solid formulation is at a concentration of about 0.002% to about 0.006% (w / w), about 0.003% to about 0.006% (w / w), about 0.004% to about 0.006% (w / w), about 0.0045% to about 0.0055% (w / w), about 0.002% to about 0.003% (w / w), about 0.003% to about 0.004% (w / w), 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).

[0068] In certain embodiments, the chelating agent is a naturally occurring compound. In certain embodiments, the chelating agent is a synthetic compound. In certain embodiments, the chelating agent is diethylenetriaminepentaacetic acid (DTPA).

[0069] 1.3.3. Surfactant In certain embodiments, the formulation comprises a surfactant.

[0070] Surfactants can lower the surface tension of liquids. In certain embodiments, the surfactant is a nonionic surfactant. Examples of surfactants include polysorbates (polyoxyethylene sorbitan monolaurate, e.g., polysorbate 20 and polysorbate 80), TRITON (t-octylphenoxypolyethoxyethanol), sodium dodecyl sulfate (SDS), sodium lauryl sulfate, sodium octyl glucoside; lauryl-, myristyl-, linoleyl-, or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl-, or stearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine; lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine (e.g., lauroamidopropyl); myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-dimethylamine; sodium cocoyl methyl taurate or disodium oleyl methyl taurate; sorbitan monopalmitate, MONAQUAT series (Mona Industries, Inc., Paterson, N.J.), polyethylene glycol (PEG), polypropylene glycol (PPG), and copolymers of polyoxyethylene and polyoxypropylene glycols (e.g., pluronic / poloxamer, PF68, etc.). In certain embodiments, the surfactant is a polysorbate. In certain embodiments, the polysorbate is polysorbate 20, polysorbate 40, polysorbate 60, or polysorbate 80.

[0071] In certain embodiments, the liquid formulation contains the 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).

[0072] In certain embodiments, the solid formulation contains 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).

[0073] 1.3.4. Polysorbate In certain embodiments, the formulation contains polysorbate.

[0074] 1.3.4.1. Polysorbate 80 In certain embodiments, the formulation contains polysorbate 80 (PS80).

[0075] In certain embodiments, the liquid formulation contains polysorbate 80 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). In certain embodiments, the formulation contains about 0.02% (w / v) of polysorbate 80.

[0076] In certain embodiments, the solid formulation contains polysorbate 80 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). In certain embodiments, the formulation contains about 0.2% (w / w) of polysorbate 80.

[0077] 1.3.4.2. Polysorbate 60 In certain embodiments, the formulation contains polysorbate 60 (PS60).

[0078] In certain embodiments, the liquid formulation comprises polysorbate 60 at 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). In certain embodiments, the formulation comprises polysorbate 60 at about 0.02% (w / v).

[0079] In certain embodiments, the solid formulation comprises polysorbate 60 at 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). In certain embodiments, the formulation comprises polysorbate 60 at about 0.2% (w / w).

[0080] 1.3.4.3. Polysorbate 40 In certain embodiments, the formulation comprises polysorbate 40 (PS40).

[0081] In certain embodiments, the liquid formulation comprises polysorbate 40 at 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). In certain embodiments, the formulation comprises polysorbate 40 at about 0.02% (w / v).

[0082] In certain embodiments, the solid formulation comprises polysorbate 40 at 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). In certain embodiments, the formulation comprises polysorbate 40 at about 0.2% (w / w).

[0083] 1.3.4.4. Polysorbate 20 In certain embodiments, the formulation comprises polysorbate 20 (PS20).

[0084] In certain embodiments, the liquid formulation comprises from about 0.01% to about 0.04%, from about 0.01% to about 0.03%, from about 0.01% to about 0.02%, from about 0.02% to about 0.03%, from about 0.03% to about 0.04%, about 0.01%, about 0.02%, about 0.03%, and / or about 0.04% (v / v) of polysorbate 20. In certain embodiments, the formulation comprises about 0.02% (v / v) of polysorbate 20.

[0085] In certain embodiments, the solid formulation comprises from about 0.1% to about 0.4%, from about 0.15% to about 0.3%, from about 0.1% to about 0.15%, from about 0.15% to about 0.2%, from about 0.2% to about 0.3%, from 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) of polysorbate 20. In certain embodiments, the formulation comprises about 0.2% (w / w) of polysorbate 20.

[0086] 1.3.5. Sugar In certain embodiments, the liquid formulation comprises sugars and their derivatives including monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, non-reducing sugars, etc. Examples of sugars include glucose, mannose, trehalose, lactose, fructose, maltose, dextran, dextrin, erythritol, glycerol, arabitol, xylitol, sorbitol, mannitol, melibiose, melezitose, raffinose, mannotriose, stachyose, maltose, lactulose, maltulose, glucitol, maltitol, lactitol, isomaltulose, etc. In certain embodiments, the sugar is a disaccharide. In certain embodiments, the disaccharide is trehalose. In certain embodiments, the disaccharide is at a concentration of about 1 to about 100 mg / mL.

[0087] 1.3.5.1. Trehalose In certain embodiments, the formulation comprises trehalose.

[0088] In certain embodiments, the liquid formulation comprises trehalose at about 100 mM to about 400 mM, about 100 mM to about 300 mM, about 150 mM to about 250 mM, about 100 mM to about 150 mM, about 150 mM to about 200 mM, about 200 mM to about 250 mM, about 250 mM to about 300 mM, about 300 mM to about 350 mM, about 350 mM to about 400 mM, about 100 mM, about 150 mM, about 200 mM, about 250 mM, about 300 mM, about 350 mM, and / or about 400 mM. In certain embodiments, the liquid formulation comprises about 200 mM of trehalose.

[0089] In certain embodiments, the solid formulation comprises about 50% to about 95%, about 60% to about 95%, about 70% to about 95%, about 80% to about 95%, about 85% to about 90%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 95% (w / w) of trehalose. In certain embodiments, the solid formulation comprises about 70% to about 80% (w / w) of trehalose. In certain embodiments, the solid formulation comprises about 75% (w / w) of trehalose.

[0090] 1.3.6. Free Amino Acids In certain embodiments, the formulation comprises at least one free amino acid. In certain embodiments, the formulation comprises only one free amino acid. In certain embodiments, the formulation comprises only two free amino acids. In certain embodiments, the formulation comprises only three free amino acids. The free amino acid can be in the L-form, D-form, or a mixture of these forms.

[0091] In certain embodiments, 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 certain embodiments, at least one free amino acid is glycine, alanine, proline, serine, aspartic acid, arginine, or lysine.

[0092] In certain embodiments, the free amino acid is in the liquid formulation at a concentration of about 25 mM to about 100 mM, about 25 mM to about 75 mM, about 40 mM to about 60 mM, about 25 mM to about 30 mM, about 30 mM to about 40 mM, about 40 mM to about 50 mM, about 50 mM to about 60 mM, about 60 mM to about 70 mM, about 70 mM to about 80 mM, about 80 mM to about 90 mM, about 90 mM to about 100 mM, about 25 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, and / or about 100 mM. In certain embodiments, the free amino acid is in the liquid formulation at a concentration of about 50 mM.

[0093] In certain embodiments, the free amino acid is in the solid formulation at a concentration of 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%, 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 certain embodiments, the free amino acid is in the solid formulation at a concentration of about 4% and about 5% (w / w). In certain embodiments, the free amino acid is in the solid formulation at a concentration of about 4.45% (w / w).

[0094] In certain embodiments, the formulation comprises alanine and at least one additional free amino acid selected from glycine, proline, serine, aspartic acid, arginine, and lysine. In certain embodiments, the formulation comprises alanine and arginine.

[0095] 1.3.6.1. Alanine In certain embodiments, the formulation comprises alanine.

[0096] In certain embodiments, the liquid formulation comprises from about 25 mM to about 100 mM, from about 25 mM to about 75 mM, from about 40 mM to about 60 mM, from about 25 mM to about 30 mM, from about 30 mM to about 40 mM, from about 40 mM to about 50 mM, from about 50 mM to about 60 mM, from about 60 mM to about 70 mM, from about 70 mM to about 80 mM, from about 80 mM to about 90 mM, from about 90 mM to about 100 mM, about 25 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, and / or about 100 mM alanine. In certain embodiments, the liquid formulation comprises about 50 mM alanine.

[0097] In certain embodiments, the solid formulation comprises from about 2% to about 8%, from about 3% to about 7%, from about 3% to about 6%, from about 4% to about 5%, from about 4.2% to about 4.8%, from about 4.4% to about 4.6%, from about 2% to about 3%, from about 3% to about 4%, from about 4% to about 4.1%, from about 4.1% to about 4.2%, from about 4.2% to about 4.3%, from about 4.3% to about 4.4%, from about 4.4% to about 4.5%, from about 4.5% to about 4.6%, from about 4.6% to about 4.7%, from about 4.7% to about 4.8%, from about 4.8% to about 4.9%, from 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) alanine (w / w). In certain embodiments, the solid formulation comprises about 4% and about 5% alanine (w / w). In certain embodiments, the solid formulation comprises about 4.45% alanine (w / w).

[0098] 1.3.6.2. Arginine In certain embodiments, the formulation comprises arginine.

[0099] In certain embodiments, the liquid formulation comprises arginine at about 25 mM to about 100 mM, about 25 mM to about 75 mM, about 40 mM to about 60 mM, about 25 mM to about 30 mM, about 30 mM to about 40 mM, about 40 mM to about 50 mM, about 50 mM to about 60 mM, about 60 mM to about 70 mM, about 70 mM to about 80 mM, about 80 mM to about 90 mM, about 90 mM to about 100 mM, about 25 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, and / or about 100 mM. In certain embodiments, the liquid formulation comprises about 50 mM of arginine.

[0100] In certain embodiments, the solid formulation comprises arginine (w / w) at 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%, 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 certain embodiments, the solid formulation comprises about 4% and about 5% of arginine (w / w). In certain embodiments, the solid formulation comprises about 4.45% of arginine (w / w).

[0101] 1.3.7. Buffer In certain embodiments, the formulation comprises at least one buffering agent (buffering component). Typically, the buffering agent, 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.

[0102] In certain embodiments, the at least one buffering agent 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-acetamido]-2-imino diacetic acid (ADA), glycylglycine, and other organic acid buffers. In certain embodiments, the buffering agent is glycylglycine, Tris, histidine, citrate, phosphate, succinate, or acetate.

[0103] In certain embodiments, the buffering component is present in the liquid formulation at a concentration of about 10 mM to about 50 mM, about 10 mM to about 40 mM, about 10 mM to about 30 mM, about 10 mM to about 15 mM, about 15 mM to about 20 mM, about 20 mM to about 25 mM, about 25 mM to about 30 mM, about 35 mM to about 40 mM, about 40 mM to about 45 mM, about 45 mM to about 50 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 40 mM, about 45 mM, and / or about 50 mM. In certain embodiments, the buffering component is present in the liquid formulation at a concentration of about 25 mM.

[0104] In certain embodiments, the buffering component is present in the solid formulation at a concentration of 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 certain embodiments, the buffering component is present in the solid formulation at a concentration of about 3% to about 4% (w / w). In certain embodiments, the buffering component is present in the solid formulation at a concentration of about 3.4% (w / w).

[0105] In certain embodiments, the buffer is glycylglycine. In certain embodiments, the formulation contains glycylglycine.

[0106] In certain embodiments, the liquid formulation contains about 10 mM to about 40 mM, about 10 mM to about 30 mM, about 10 mM to about 15 mM, about 15 mM to about 20 mM, about 20 mM to about 25 mM, about 25 mM to about 30 mM, about 35 mM to about 40 mM, about 40 mM to about 45 mM, about 45 mM to about 50 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, and / or about 50 mM of glycylglycine. In certain embodiments, the liquid formulation contains about 25 mM of glycylglycine.

[0107] In certain embodiments, the solid preparation contains glycylglycine at 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 certain embodiments, the solid preparation contains glycylglycine at about 3% to about 4% (w / w). In certain embodiments, the solid preparation contains glycylglycine at about 3.4% (w / w).

[0108] 1.3.7.1. pH In certain embodiments, the liquid preparation has a pH of 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 certain embodiments, the preparation has a pH of about 7.5.

[0109] 1.3.8. Stabilization In certain embodiments, the liquid formulation stabilizes the IL12-ABD fusion protein against heat stress. In certain embodiments, the liquid formulation reduces the formation of invisible particles as measured by FlowCam after 14 days of heat stress at 40°C. In certain embodiments, the liquid formulation reduces the formation of high molecular weight (HMW) species as measured by size exclusion high performance liquid chromatography (SEC-HPLC) after 14 days of heat stress at 40°C. In certain embodiments, the liquid formulation reduces the formation of low molecular weight (LMW) species as measured by size exclusion high performance liquid chromatography (SEC-HPLC) after 14 days of heat stress at 40°C.

[0110] In certain embodiments, the liquid formulation stabilizes the IL12-ABD fusion protein against freeze-thaw stress. In certain embodiments, the liquid formulation reduces the formation of invisible particles as measured by FlowCam after freeze-thaw stress by 5 freeze-thaw cycles. In certain embodiments, the liquid formulation reduces the formation of high molecular weight (HMW) species as measured by size exclusion high performance liquid chromatography (SEC-HPLC) after freeze-thaw stress by 5 freeze-thaw cycles. In certain embodiments, the liquid formulation reduces the formation of low molecular weight (LMW) species as measured by size exclusion high performance liquid chromatography (SEC-HPLC) after freeze-thaw stress by 5 freeze-thaw cycles.

[0111] In certain embodiments, the liquid formulation stabilizes the IL12-ABD fusion protein from shear stress. In certain embodiments, the liquid formulation reduces the formation of visible particles as measured by FlowCam after 24 hours of simulated shear stress at 900 RPM. In certain embodiments, the liquid formulation reduces the formation of high molecular weight (HMW) species as measured by size exclusion high performance liquid chromatography (SEC-HPLC) after 24 hours of simulated shear stress at 900 RPM. In certain embodiments, the liquid formulation reduces the formation of low molecular weight (LMW) species as measured by size exclusion high performance liquid chromatography (SEC-HPLC) after 24 hours of simulated shear stress at 900 RPM.

[0112] 1.4. Administration of the formulation Suitable routes of administration of the IL12-ABD fusion protein formulations described herein include, but are not limited to, parenteral (e.g., by subcutaneous, intravenous, intramuscular, intradermal, or intrasternal injection or infusion, such as a sterile injectable aqueous or non-aqueous solution or suspension). 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.

[0113] In some embodiments, the formulation is suitable for administration in single or multiple doses.

[0114] 1.5. Dosage form 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.

[0115] In various embodiments, the dosage form is a pre-filled syringe. In various embodiments, the dosage form is an auto-injector pen.

[0116] In various embodiments, the dosage form includes one or more unit doses of the formulations described herein. In typical embodiments, the formulation is a liquid formulation. In other embodiments, the formulation is a dry formulation including, but not limited to, a lyophilized product. In certain embodiments, the dosage form includes a dry formulation and a certain amount of aqueous diluent.

[0117] In some embodiments, the unit dosage form includes a formulation comprising an IL12-ABD fusion protein at about 10 μg / mL to about 50 μg / mL, alanine at about 25 mM to about 100 mM, trehalose at about 100 mM to about 400 mM, glycylglycine at about 10 mM to about 50 mM, polysorbate 20 at about 0.01% to about 0.04% (v / v), and diethylenetriaminepentaacetic acid (DTPA) at about 5 μM to about 20 μM, and the formulation has a pH of about 6.8 to about 8.0.

[0118] In some embodiments, the unit dosage form includes a formulation comprising an IL12-ABD fusion protein at about 0.01% to about 0.03% (w / w), alanine at about 2% to about 8% (w / w), trehalose at about 50% to about 95% (w / w), glycylglycine at about 2% to about 8% (w / w), polysorbate 20 at about 0.1% to about 0.4% (w / w), and diethylenetriaminepentaacetic acid (DTPA) at about 0.002% to about 0.006% (w / w).

[0119] 1.6. Treatment methods In another aspect, provided herein is a method of treating a disease or disorder in a patient, the method comprising administering to the patient an IL12-ABD fusion protein formulation described herein. In some embodiments, the patient has an IL-12 related disease or disorder, such as cancer, an autoimmune disease, or an infectious disease. In some embodiments, the IL12-ABD fusion protein is administered as an immune adjuvant for the treatment of cancer, an autoimmune disease, or an infectious disease, such as administering the IL12-ABD fusion protein together with a vaccine antigen.

[0120] In another aspect, provided herein is a method of inhibiting tumor growth in a patient, the method comprising administering to the patient an IL12-ABD fusion protein formulation described herein.

[0121] In another aspect, provided herein is a method of treating cancer in a patient, the method comprising administering to the patient an IL12-ABD fusion protein formulation described herein.

[0122] In certain embodiments, cancers to be treated include, but are not limited to, carcinomas, granulomas, sarcomas, and certain leukemias or lymphoid malignancies. More specific examples of such cancers include squamous cell cancer (e.g., epithelial squamous cell cancer), lung cancer including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung, peritoneal cancer, hepatocellular cancer, gastric cancer or stomach cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, liver cancer, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, skin cancer / melanoma, and head and neck cancer, and metastases associated with any of the primary tumors.

[0123] In another aspect, provided herein is a method of treating an autoimmune disease in a patient, the method comprising administering to the patient an IL12-ABD fusion protein formulation described herein.

[0124] In certain embodiments, autoimmune diseases to be treated include, but are not limited to, rheumatoid arthritis, juvenile idiopathic arthritis, and graft-versus-host disease (GvHD).

[0125] In another aspect, provided herein is a method of treating an infectious disease in a patient, the method comprising administering to the patient an IL12-ABD fusion protein formulation described herein.

[0126] In certain embodiments, infectious diseases to be treated include, but are not limited to, local or systemic viral infections such as encephalitis, influenza, colds, immunodeficiency, and herpes virus infections. In certain embodiments, the infectious disease is caused by bacteria, viruses, protozoa, helminths, or other microbial pathogens. In certain embodiments, the infectious disease to be treated is caused by hepatitis virus, human immunodeficiency virus (HIV), human T-lymphotropic virus (HTLV), herpes virus, Epstein-Barr virus, or human papillomavirus.

[0127] The following are examples of specific embodiments for carrying out the present invention. The examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way. Although efforts have been made to ensure accuracy with respect to the numbers used (e.g., amounts, temperatures, etc.), some experimental errors and deviations are, of course, to be tolerated.

Example

[0128] Example 1: Stability study of formulations with various buffers and pH Formulations of the IL12-ABD fusion protein SONE-IL12-ABD (SEQ ID NO: 11) were prepared as shown in Table 1.

Table 1

[0129] 1.1. Heat stress study The formulations were subjected to heat stress at 40 °C for 7 days and monitored for osmolality, pH, A280, right angle light scattering (RALS), DLS, Flowcam, SEC-HPLC, and reduced and non-reduced SDS-PAGE. The results of osmolality, pH, A280, and RALS are shown in Table 2.

Table 2

[0130] As shown in Table 2, the concentration after incubation at 40 °C for 7 days increased most in the formulations containing acetate and citrate. In addition, when evaluated using RALS, formulations with a higher pH and those 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 those containing succinate (having two carboxyl groups).

[0131] The formulations were evaluated by DLS after incubation at 40 °C for 7 days. As shown in Table 3, the diameter of the particle size increased in the formulations with a pH less than 7 after heat stress. [Table 3]

[0132] The formulations were evaluated for the presence of sub-particle substances by Flowcam after incubation at 40 °C for 7 days. The results are shown in Table 4. [Table 4]

[0133] The formulations were heat-stressed at 40 °C for 7 days, 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 5 and 6. At T7 after heat stress, formulations with a pH < 7.0 showed a higher % of HMW species and a lower % of main recovery. Formulations with a higher pH > 7.0 (A1 - A4 and A-CF) had the least amount of aggregates and were stable at T7 against heat stress. [Table 5] [Table 6]

[0134] The formulations subjected to heat stress at 40 °C for 7 days were also analyzed by reducing and non-reducing SDS-PAGE. Each formulation matched A-CF on the reducing gel. The non-reducing gels showed more variability and extra HMW bands in A5 and A6 containing histidine with and without PS20, A7 and A8 containing water with and without PS20, A9 and A10 containing acetate with and without PS20, and A11 and A12 containing citrate with and without PS20.

[0135] 1.2. Freeze-thaw studies The formulations at T0 were subjected to 5 freeze-thaw cycles and then analyzed for osmolality, pH, RALS, A280, DLS, SEC-HPLC, and reducing and non-reducing SDS-page. The results of osmolality, pH, A280, and right angle light scattering (RALS) are shown in Table 7.

Table 7

[0136] As shown in Table 7, after the freeze-thaw cycles, A7 and A8 containing water with and without PS20, A9 containing acetate with PS20, and A14 containing succinate had low recovery percentages of less than 90% with respect to concentration. Additionally, when evaluated using RALS, this trend was consistent with the temperature stress with the smallest RALS values for the formulations with higher pH, along with the formulations containing citrate.

[0137] The formulations were evaluated by DLS after incubation following 5 freeze-thaw cycles. As shown in Table 8, the polydispersity index (PDI) increased in all formulations except A-CF after 5 freeze-thaws.

Table 8

[0138] The formulation was subjected to 5 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 formulation is freeze-thaw sensitive. Consistent with the effects of time and temperature (thermal stress study), formulations at higher pH had relatively higher recovery % for both the main peak and total peaks, and there was less increase in LMW species.

Table 9

Table 10

[0139] Formulations subjected to 5 freeze-thaw cycles were also analyzed by reducing and non-reducing SDS-PAGE. Each formulation matched A-CF on both the reducing and non-reducing gels.

[0140] 1.3. Simulated shear stress study The formulation at T0 was subjected to simulated shear stress at 900 RPM for 24 hours at room temperature and analyzed for osmolality, pH, RALS, DLS, SEC, and SDS-PAGE. The results for osmolality, pH, and RALS are shown in Table 11.

Table 11

[0141] Consistent with the results obtained for heat and freeze-thaw stress, formulations at pH higher than 7.0 showed relatively less light scattering after simulated shear stress, as shown in Table 11. The data also demonstrate that the SONE-IL12-ABD formulation is susceptible to the effects of simulated shear stress. All formulations above pH 6.0 remained clear during simulated shear stress, except for the formulation containing phosphate without PS20 at pH 7.0.

[0142] The formulations were evaluated by DLS after incubation following simulated shear stress. As shown in Table 12, all formulations had a significant increase in the diameter of the particle size after simulated shear stress, but the formulations with pH > 7.0 had relatively smaller particle sizes compared to the other formulations.

Table 12

[0143] The formulations were subjected to simulated shear stress at 900 RPM for 24 hours, 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 13 and 14. Aggregates were more in non-PS20 formulation A10 containing acetate and A12 containing citrate. The highest recovery after simulated shear stress was shown by the formulations containing Tris.

Table 13

Table 14

[0144] The formulations subjected to simulated shear stress were also analyzed by reducing and non-reducing SDS-PAGE. Each formulation, except for formulation A12 containing citrate after shear stress, matched A-CF on the reducing gel. The non-reducing gel showed more variability and increased degradation in formulation A3 containing Tris with PS20, and HMW streaking in some samples after shear stress.

[0145] The results demonstrated that SONE-IL12-ABD formulations containing only buffer with and without PS20 were sensitive to heat, freeze-thaw, and shear. The stability of the formulations was pH-dependent, and formulations with a pH of 7.0 or higher showed relatively higher stability when analyzed after heat stress, freeze-thaw stress, and simulated shear stress. Formulations containing phosphate and tris with and without PS20 were relatively more stable compared to other formulations under physical and heat stress. Examples of such formulations included 10 mM tris at pH 7.5 with 0.02% PS20.

[0146] Example 2: Stability study of formulations with various ionic strengths Formulations of SONE-IL12-ABD were prepared as shown in Table 15 using an approximate SONE-IL12-ABD concentration of 0.050 mg / ml. [Table 15]

[0147] 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. [Table 16]

[0148] Tm was highest in samples with a high salt content and B-CF. Tagg was highest in salt-free samples (B1 without NaCl, B6 with tris, B7 with histidine, and B-CF).

[0149] 2.1. Heat stress study The formulations were subjected to heat stress at 40°C for 14 days, and the osmolality, pH, A280, RALS, Flowcam, SEC-HPLC, and reduced and non-reduced SDS-page were monitored. The results of osmolality, pH, A280, and RALS are shown in Tables 17 and 18. [Table 17]

Table 18

[0150] As shown in Table 18, the percent recovery at T14 was low for Formulations B2 and B3 containing incremental amounts of salt. Formulation B5, which contains a high amount of salt, had a high recovery, similar to Formulations B6 and B-CF containing Tris. B1, which contains no salt, B2, which contains 10 mM NaCl, and B7, which contains histidine, had a recovery of 85% - 105%.

[0151] The formulations were evaluated for the presence of subparticulate matter by Flowcam after incubation at 40 °C for 7 and 14 days. The results are shown in Table 19.

Table 19

[0152] The formulations were heat stressed at 40 °C for 14 days, 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 20 - 22. B3, which contains 50 mM NaCl, had the lowest amount of LMW% at T14. Formulations containing Tris and histidine (B6 and B7) had a significant increase in HMW and LMW. The formulation containing histidine (B7) had the highest total recovery at T14.

Table 20

Table 21

Table 22

[0153] The formulations subjected to heat stress 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 containing 10 mM of NaCl, B6 containing Tris, and B7 containing histidine had large HMW bands. B1 without salt, B6 containing Tris, B7 containing histidine, and B-CF also had high concentrations of LMW bands.

[0154] 2.2. Freeze-thaw studies The formulations at T0 were subjected to 5 freeze-thaw cycles and then analyzed for osmolality, pH, RALS, A280, Flowcam, SEC-HPLC, and reducing and non-reducing SDS-page. The results of osmolality, pH, A280, and RALS are shown in Table 23. [Table 23]

[0155] As shown in Table 23, after the freeze-thaw cycle, formulation B3 containing 50 mM of NaCl had a low recovery for the concentration after freeze-thaw. B5 with a high salt content, B6 containing Tris, and B-CF had recoveries exceeding 100%. Furthermore, the RALS value after freeze-thaw slightly increased for formulation B6 containing Tris.

[0156] The formulations were evaluated for the presence of sub-particle matter by Flowcam after 5 freeze-thaw cycles. The results are shown in Table 24. [Table 24]

[0157] The formulations were subjected to 5 freeze-thaw cycles, 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 25 and 26. Formulation B5, which contained a large amount of salt, had no LMW after freeze-thaw. All of B6, which contained Tris but no salt, B7, which contained histidine, and B-CF had the highest total recovery after freeze-thaw.

Table 25

Table 26

[0158] The formulations subjected to 5 freeze-thaw cycles were also analyzed by reducing and non-reducing SDS-PAGE. No significant difference was observed by SDS-PAGE after 5 freeze-thaw cycles.

[0159] 2.3. Simulated shear stress study The formulations at T0 were subjected to simulated shear stress at 900 RPM at room temperature for 24 hours and analyzed for osmolality, pH, RALS, DLS, SEC, and SDS-PAGE. The results of osmolality, pH, and RALS are shown in Table 27.

Table 27

[0160] As shown in Table 27, the RALS values increased for all formulations, especially for the formulations containing 50 mM NaCl, 100 mM NaCl, and histidine (B3, B4, and B7).

[0161] The formulations were evaluated by DLS after incubation following simulated shear stress. As shown in Table 28, all formulations had large particle size diameters after simulated shear stress. Since the concentration of the samples was approximately 0.050 mg / ml, dynamic light scattering was not observed at this low concentration, but shear stress increased the particle size and thus made it measurable by DLS. This was used as a screening tool for formulations subjected to simulated shear stress. The data suggest that formulations containing 0 mM or 10 mM NaCl (D1 and D2) showed low light scattering measured by DLS and had smaller particle sizes compared to other formulations.

Table 28

[0162] The formulations were subjected to simulated shear stress at 900 RPM for 24 hours, and 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 29 and 30. Formulations B5 and B-CF containing a lot of salt had the highest main % after simulated shear stress. Formulations B5 and CF containing a lot of salt also had very low HMW and LMW compared to other formulations. Salt-free formulations 1 and CF had the highest total recovery exceeding 90%.

Table 29

Table 30

[0163] The formulations subjected to simulated shear stress were also analyzed by reducing and non-reducing SDS-PAGE. More aggregates were observed in the non-reducing gel compared to B-CF. Fragmentation was observed in B2 containing 10 mM NaCl and B3 containing 50 mM NaCl.

[0164] The results demonstrated that in the presence of NaCl, the SONE-IL12-ABD formulation was more susceptible to the effects of simulated shear stress. The Tm measurement values were lower for formulations with less salt at T0, and the Tagg measurement values were highest for the formulation without added salt at pH 7.5. The freeze-thaw studies showed similar susceptibilities among the formulations, except for B-CF. Furthermore, the temperature stress studies demonstrated that the salt-free formulation had a relatively higher total recovery compared to the salt-containing formulations.

[0165] Example 3: Stability Studies of Formulations Containing Various Surfactants Formulations of SONE-IL12-ABD were prepared as shown in Table 31 using an approximate SONE-IL12-ABD concentration of 0.050 mg / ml. [Table 31]

[0166] The formulations were subjected to temperature-ramping RALS and IF to obtain the Tagg and Tm for each formulation. The Tm and Tagg values are shown in Table 32. [Table 32]

[0167] Formulation C6 (0.05 mg / ml SONE-IL12-ABD, 25 mM glygly, 0.02% PS20, 10 μM DTPA) showed high values for both Tm and Tagg at 54 °C and 70 °C, respectively.

[0168] 3.1. Thermal Stress Studies The formulations were subjected to thermal stress at 40 °C for 14 days and monitored for osmolality, pH, A280, RALS, Flowcam, SEC-HPLC, and reduced and non-reduced SDS-page. The results of osmolality, pH, A280, and RALS are shown in Tables 33 and 34. [Table 33]

Table 34

[0169] As shown in Table 33, during the 14-day incubation, the pH increased slightly. Formulations C5 and C-CF containing PEG3350 had the highest recovery percentage of approximately 90% at the concentration based on A280 at T14.

[0170] After incubating the formulations at 40 °C for 7 and 14 days, the presence of sub-particle matter was evaluated by Flowcam. As shown in Table 35, the number of invisible particles increased for each formulation at T7 and T14, and the particles increased the most in C4 containing poloxamer 188.

Table 35

[0171] The formulations were subjected to heat stress at 40 °C for 14 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 36 - 38. After 14 days of incubation, all formulations except C-CF showed a significant increase in HMW%. The total recovery at T14 ranged from 88% of formulation C2 (containing 25 mM glygly, 0.02% PS20) to 96% of formulation C6 (containing 25 mM glygly, 0.02% PS20, and 10 μM DTPA). C-CF had a recovery percentage of 103%.

Table 36

Table 37

Table 38

[0172] The formulations subjected to heat stress at 40 °C for 14 days were also analyzed by reducing and non-reducing SDS-PAGE. The T0 sample did not show HMW on the non-reducing gel, and the non-reducing gel showed more variability associated with increased degradation. C6 (containing PS20 and DTPA) showed 100% main band on the non-reducing gel at T14.

[0173] 3.2. Freeze-thaw studies The formulations at T0 were subjected to 5 freeze-thaw cycles and then analyzed for osmolality, pH, RALS, A280, Flowcam, SEC-HPLC, and reducing and non-reducing SDS-page. The results of osmolality, pH, A280, and RALS are shown in Table 39.

Table 39

[0174] As shown in Table 39, except for C2 (25 mM glygly, 0.02% PS20) with 101% recovery, the concentration decreased slightly after freeze-thaw.

[0175] The formulations were evaluated for the presence of sub-particle material by Flowcam after 5 freeze-thaw cycles. The results are shown in Table 40. Invisible particles increased in each formulation after freeze-thaw. The particles increased the most in C6 (25 mM glygly, 0.02% PS20, 10 μM DTPA).

Table 40

[0176] The formulation was subjected to 5 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 41 and 42. C3 containing PS80, C6 containing PS20 and DTPA, and C-CF had 100% recovery by SEC after freeze-thaw. HMW increased slightly in all formulations except C1 containing glygly. Each formulation had a main peak percent exceeding 94%. C1 containing glygly had the lowest amount of LMW after freeze-thaw.

Table 41

Table 42

[0177] The formulations subjected to 5 freeze-thaw cycles were also analyzed by reducing and non-reducing SDS-PAGE. In the reducing gel after freeze-thaw, the LMW% increased slightly in each formulation. No change was seen in the non-reducing gel, which showed a 100% main peak.

[0178] 3.3. Simulated shear stress study The formulation at T0 was subjected to simulated shear stress at 900 RPM at room temperature for 24 hours and analyzed for osmolality, pH, A280, RALS, SEC, and SDS-PAGE. The results of osmolality, pH, A280, and RALS are shown in Table 43.

Table 43

[0179] As shown in Table 43, the RALS values increased significantly after simulated shear stress for the formulation containing glyceryl (C1), the formulation containing PS80 (C3), and C-CF, indicating that these formulations were relatively more sensitive to shear stress. C6, which contains 25 mM glyceryl, 0.02% PS20, and 10 μM DPTA, had the smallest RALS value, indicating that this formulation was relatively less sensitive to shear stress. The formulations containing PS20, PS80, and poloxamer (C2, C3, and C4) each had a turbid appearance after simulated shear stress.

[0180] The formulations were subjected to simulated shear stress at 900 RPM for 24 hours, 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 44 and 45. The results demonstrated that C6 (25 mM glyceryl, 0.02% PS20, 10 μM DTPA) had a relatively higher total area recovery compared to the other formulations.

Table 44

Table 45

[0181] The formulations subjected to simulated shear stress were also analyzed by reducing and non-reducing SDS-PAGE. The reducing gel showed no significant differences after shear, while the non-reducing gel showed an increase in HMW. C6 (25 mM glyceryl, 0.02% PS20, 10 μM DTPA) had the smallest amount of HMW in the gel after shear.

[0182] SDS-PAGE and SEC data after heat stress, and RALS and SDS-PAGE data after simulated shear stress demonstrated that PS20 improved stability. The total recovery data of SEC after heat stress demonstrated that C6 (containing PS20 and DTPA) and C-CF were relatively more stable among the formulations. Furthermore, tests after freeze-thaw stress demonstrated that C2 (containing PS20, C3 containing PS80, C6 containing PS20 and DTPA, and C-CF) were relatively more stable against freeze-thaw stress compared to other formulations.

[0183] Example 4: Stability Study of Formulations Containing Various Amino Acid Stabilizers Formulations of SONE-IL12-ABD were prepared as shown in Table 46 using an SONE-IL12-ABD concentration of approximately 20 μg / ml. [Table 46]

[0184] 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 46A. [Table 47]

[0185] 4.1. Heat Stress Study The formulations were subjected to heat stress at 40 °C for 14 days, and osmolality, pH, A280, RALS, Flowcam, SEC-HPLC, and reduced and non-reduced SDS-page were monitored. The results of osmolality, pH, A280, and RALS are shown in Tables 47 and 48. [Table 48] [Table 49]

[0186] As shown in Table 48, except for formulation D2 (containing serine), the concentration decreased in each formulation after 14 days of incubation.

[0187] The formulations were evaluated for the presence of sub-particle substances by Flowcam after incubation at 40 °C for 7 and 14 days. As shown in Table 49, except for formulation D5 (containing arginine), the number of invisible particles at T14 increased in most formulations. The number of invisible particles of formulation D7 (containing proline) at T14 increased significantly. [Table 50]

[0188] The formulations were subjected to heat stress 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 - 52. Formulations D5 (containing arginine) and D6 (containing lysine) had the highest main peak percentages by SEC. HMW increased at T7 and T14, and showed a smaller increase for D5 (containing arginine) and D6 (containing lysine). [Table 51] [Table 52] [Table 53]

[0189] The formulations subjected to heat stress 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% main band without HMW and LMW. Each formulation behaved similarly at T7 and T14. Some HMW was seen in the reducing gel, while some LMW was present in the non-reducing gel. [Table 54]

Table 55

[0190] 4.2. Freeze - thaw study The formulation at T0 was subjected to 5 freeze - thaw cycles and then analyzed for osmolality, pH, RALS, A280, Flowcam, SEC - HPLC, and reduced and non - reduced SDS - page. The results of osmolality, pH, A280, and RALS are shown in Table 55.

Table 56

[0191] As shown in Table 55, except for formulations D2 (containing serine) and D7 (containing proline), the concentration decreased slightly after freeze - thaw.

[0192] The formulations were evaluated for the presence of sub - particulate matter by Flowcam after 5 freeze - thaw cycles. The results are shown in Table 56. Sub - visible particles increased in most formulations.

Table 57

[0193] The formulations were subjected to 5 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 57 and 58. Formulation D7 (containing proline) showed relatively higher HMW and was sensitive to repeated freeze - thaw. SEC data indicate that formulations containing arginine, lysine, serine, alanine, and glycine are relatively insensitive to repeated freeze - thaw and show nearly 100% recovery at T14.

Table 58

Table 59

[0194] The formulations subjected to 5 freeze - thaw cycles were also analyzed by reducing and non - reducing SDS - PAGE. The results are shown in Table 59. Some LMW and HMW were observed after freeze - thaw, for example, in formulation D7 (containing proline).

Table 60

[0195] 4.3. Simulated shear stress study The formulations at T0 were subjected to simulated shear stress at 900 RPM for 24 hours at room temperature and analyzed for osmolality, pH, A280, RALS, SEC, and SDS - PAGE. The results of osmolality, pH, A280, and RALS are shown in Tables 60 and 61.

Table 61

[0196] As shown in Table 60, the concentration increased after simulated shear stress, especially in formulation D2 (containing serine).

Table 62

[0197] As shown in Table 61, the RALS values increased across all formulations after simulated shear stress. The formulations containing alanine and proline (D3 and D7) had the smallest RALS values and showed improved stability during shear.

[0198] The formulation was subjected to simulated shear stress at 900 RPM for 24 hours, 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 62 and 63. The results indicated that formulations containing serine and alanine (D2 and D3) showed higher total recovery after simulated shear stress and were relatively less sensitive to shear stress.

Table 63

Table 64

[0199] The formulations subjected to simulated shear stress were also analyzed by reducing and non-reducing SDS-PAGE. The results are shown in Table 64. HMW and LMW were observed in all formulations during shear stress on the gel. Formulations containing arginine and proline (D5 and D7) showed relatively lower percentages of HMW and LMW during shear stress, indicating improved stability.

Table 65

[0200] Based on SEC data using heat stress, 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 most amount of LMW during simulated shear stress, indicating relatively higher sensitivity to shear stress. Furthermore, the results suggest that formulations containing alanine and proline (D3 and D7) protect from agitation / simulated shear stress, while formulations containing arginine and lysine (D5 and D6) protect from heat stress.

[0201] Example 5: Stability Study of Formulations Containing Various Carbohydrate Stabilizers, Cryoprotectants, and / or Lyoprotectants Formulations of SONE-IL12-ABD were prepared as shown in Table 65 using an SONE-IL12-ABD concentration of approximately 20 μg / ml. [Table 66]

[0202] The formulations shown in Table 65 were also lyophilized according to the lyophilization cycle shown in Table 66. [Table 67]

[0203] 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 67. The Tm and Tagg are similar across all formulations. Formulations containing arginine and trehalose (E6), and alanine, arginine, and trehalose (E8) have the highest Tm. [Table 68]

[0204] 5.1. Thermal Stress Study The liquid and lyophilized formulations were subjected to thermal stress at 40 °C for 14 days and monitored for visual appearance, osmolality, pH, A280, RALS, Flowcam, SEC-HPLC, reduced and non-reduced SDS-page, and ELISA. The results of visual appearance are shown in Table 68, osmolality, pH, and RALS in Tables 69 and 70, and A280 in Tables 71 and 72.

[0205] As shown in Table 68, all liquids and reconstituted samples were transparent, colorless, and had no visible particles. The lyophilized cakes of the formulation containing mannitol (E4) had no cracks or peeling from the glass. The lyophilized cakes of the formulation containing E-CF, alanine, sucrose, and mannitol (E5) completely disintegrated at T14. [Table 69] [Table 70] [Table 71] [Table 72] [Table 73]

[0206] As shown in Table 70, for the lyophilized formulations containing alanine and sucrose (E3), alanine and mannitol (E4), alanine, sucrose, and mannitol (E5), and arginine and trehalose (E6), the RALS increased slightly.

[0207] The formulations were evaluated for the presence of sub-particle matter by Flowcam after incubation at 40 °C for 7 and 14 days. As shown in Table 73, the number of invisible particles increased in all liquid formulations after 14 days of incubation, and the formulation containing proline (E7) had a relatively higher number of particles. As shown in Table 74, the number of invisible particles increased in all lyophilized formulations after 14 days of incubation, and the formulation containing proline and trehalose (E7) had a relatively higher number of particles. [Table 74]

Table 75

[0208] The formulation was subjected to heat stress 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.

[0209] Liquid formulations containing arginine and trehalose (E6), and liquid formulations containing alanine, arginine, and trehalose (E8) showed no change in HMW% by SEC after 14 days of incubation. The results demonstrated that the presence of arginine protects the liquid formulation from heat stress. The LMW% was the lowest in the liquid formulation containing alanine, sucrose, and mannitol (E5). The main peak% at T14 was the highest in the liquid formulation containing arginine and trehalose (E6), and the liquid formulation containing alanine, arginine, and trehalose (E8). The results also demonstrated that the total recovery of the liquid formulation containing alanine and trehalose (E2), and the liquid formulation containing alanine, arginine, and trehalose (E8) was relatively higher.

[0210] The lyophilized formulation was relatively more stable at 40 °C for 14 days with respect to total recovery and main peak recovery. The HMW% was slightly higher in E-CF, and the main peak recovery and LMW% at T14 were relatively lower in the formulation containing arginine and trehalose (E6).

Table 76

Table 77

Table 78

Table 79

Table 80

Table 81

[0211] The liquid and lyophilized formulations E1 (trehalose), E2 (alanine and trehalose), E4 (alanine and mannitol), and E8 (alanine, arginine, and trehalose) that were 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) exhibited the fewest impurities.

[0212] The 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 shown in Table 81.

Table 82

[0213] 5.2. Freeze-Thaw Studies The formulations at T0 were subjected to 5 freeze-thaw cycles and then analyzed for osmolality, pH, RALS, A280, Flowcam, and SEC-HPLC. The results for osmolality, pH, A280, and RALS are shown in Table 81A.

Table 83

[0214] As shown in Table 81A, with the exception of formulation E-CF, the concentrations decreased slightly after freeze-thaw.

[0215] The formulations were evaluated for the presence of sub-particle substances by Flowcam after 5 freeze-thaw cycles. The results are shown in Table 82. Sub-visible particles in the range of 2 - 10 μm increased in all formulations.

Table 84

[0216] The formulations were subjected to 5 freeze-thaws, and total recovery, fusion protein recovery, and percent impurities consisting of HMW and LMW were monitored by SEC-HPLC. The results are shown in Tables 83 and 84. HMW% was similar across all formulations, except for the formulation containing trehalose (E1) which had relatively lower HMW species after E-CF and freeze-thaw. The formulations containing alanine and sucrose (E3), and alanine, sucrose, and mannitol (E5) showed the lowest amount of LMW%. Formulation E6 containing alanine, arginine, and trehalose showed the lowest recovery % after freeze-thaw.

Table 85

Table 86

[0217] 5.3. Simulated Shear Stress Study The formulations at T0 were subjected to simulated shear stress at 900 RPM at room temperature for 24 hours and analyzed for osmolality, pH, A280, RALS, SEC, and DLS. The results for osmolality, pH, A280, and RALS are shown in Table 85.

Table 87

[0218] As shown in Table 85, formulation E1 containing trehalose and E2 containing alanine and trehalose had the lowest concentration after simulated shear stress, suggesting that there were fewer light-scattering particles contributing to or interfering with the absorbance measurement at A280. Furthermore, formulation E2 containing alanine and trehalose had better resistance to shear stress with the smallest RALS value. Formulations E6 containing arginine and trehalose and E8 containing alanine, arginine, and trehalose were relatively more sensitive to shear stress compared to other formulations, as indicated by the larger RALS values.

[0219] The formulations were subjected to simulated shear stress at 900 RPM for 24 hours, 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 86 and 87. The HMW% increased in all liquid formulations after shear when monitored by SEC. The LMW% increased significantly in formulation E1 containing trehalose and formulation E8 containing alanine, arginine, and trehalose. Formulation E7 containing proline and trehalose also had the highest main peak%, and E2 containing alanine and trehalose showed the highest total recovery% after shear, suggesting that it was relatively less sensitive to shear stress.

Table 88

Table 89

[0220] 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 a relatively small particle size diameter of approximately 1.2 nm after simulated shear stress.

Table 90

[0221] In summary, the results of Example 5 demonstrated that arginine protects against heat stress and alanine protects against shear stress. For example, Formulations E8 (containing alanine, arginine, and trehalose) and E2 (containing alanine and trehalose) stabilized SONE-IL12-ABD, and Liquid E2 and E8 formulations had the highest total recovery percentages by SEC after 14 days of incubation.

[0222] Example 6: Stability Study of Formulations Containing Various Stabilizers Formulations of SONE-IL12-ABD were prepared as shown in Table 89 using an approximate SONE-IL12-ABD concentration of 20 μg / ml.

Table 91

[0223] The formulations shown in Table 89 were also lyophilized according to the lyophilization cycle shown in Table 66.

[0224] The liquid formulations were subjected to temperature-ramping RALS and IF to obtain Tagg and Tm for each formulation. The Tm and Tagg values are shown in Table 90. Tm and Tagg were similar across all formulations.

Table 92

[0225] 6.1. Heat Stress Study Liquid and lyophilized formulations were subjected to heat stress at 40 °C for 14 days and monitored for visual appearance, osmolality, pH, A280, RALS, Flowcam, SEC-HPLC, reduced and non-reduced SDS-page, and ELISA. The results of visual appearance are shown in Table 91, osmolality, pH, and RALS in Tables 92 and 93, and A280 in Tables 94 and 95.

[0226] As shown in Table 91, all liquids and reconstituted samples were transparent, colorless, and had no visible particles. The T7 F4 vial containing more trehalose than mannitol had an imperfect stopper that prevented lyophilization, but the T14 data for F4 could be measured. F6, which contained more mannitol than trehalose, had a lyo cake that completely disintegrated at T0 and did not change at T14. The cakes of the lyophilized formulations containing the indicated alanine and arginine were improved compared to the cakes of the lyophilized formulations containing mannitol and trehalose. [Table 93] [Table 94] [Table 95] [Table 96] [Table 97]

[0227] As shown in Table 93, the osmolality increased more in F1, which contained more alanine than arginine, and in F2, which contained equal amounts of alanine and arginine, in the T14 lyo samples.

[0228] As shown in Table 94, the concentration decreased in all liquid formulations except F1, which contained more alanine than arginine.

[0229] The formulations were evaluated for the presence of sub-particle substances by Flowcam after incubation at 40 °C for 7 and 14 days. As shown in Table 96, the number of invisible particles increased in all liquid formulations after 14 days of incubation, and formulation F6, which contains more mannitol than trehalose, had the greatest increase. As shown in Table 97, the number of invisible particles increased in all freeze-dried formulations after 14 days of incubation. [Table 98] [Table 99]

[0230] The formulations were subjected to heat stress at 40 °C for 14 days, and 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 98 - 103.

[0231] Liquid formulation F5, which contains the same amount of trehalose and mannitol, had the least amount of LMW by SEC at T14. Liquid formulations containing arginine had a very low amount of HMW at T14. The main peak percentage was the highest in the formulations containing arginine. The results also demonstrated that the formulations containing arginine were protected from heat stress.

[0232] The LMW% decreased in all freeze-dried formulations by SEC at T14. All freeze-dried formulations had a very low HMW% at T14. The main peak percentage was relatively similar across all freeze-dried formulations, and F-LF and F5 (containing the same amount of trehalose and mannitol) were the highest at 96%. [Table 100] [Table 101] [Table 102]

Table 103

Table 104

Table 105

[0233] The liquid and lyophilized formulations F-LF, F1, F2, and F3, which were 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 that the formulation containing F-LF and mannitol showed the fewest impurities.

[0234] The liquid and lyophilized formulations F-LF, F4, F5, and F6 were also analyzed by ELISA. The results are shown in Table 104.

Table 106

[0235] 6.2. Freeze-Thaw Studies The formulations at T0 were subjected to 5 freeze-thaw cycles and then analyzed for osmolality, pH, RALS, A280, Flowcam, and SEC-HPLC. The results of osmolality, pH, A280, and RALS are shown in Table 105.

Table 107

[0236] The formulations were evaluated for the presence of sub-particle matter by Flowcam after 5 freeze-thaw cycles. The results are shown in Table 106. Sub-visible particles in the range of 2 - 10 μm increased in all formulations.

Table 108

[0237] The formulations were subjected to 5 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 107 and 108. The LMW% decreased significantly in F1 by SEC after freeze - thaw. The main% after freeze - thaw was consistent at about 93% for all formulations. F - LF containing alanine and trehalose, and F4 containing more trehalose than mannitol had the highest total recovery percentage at 90% after freeze - thaw. [Table 109] [Table 110]

[0238] 6.3. Simulated shear stress study The formulations at T0 were subjected to simulated shear stress at 900 RPM at room temperature for 24 hours and analyzed for osmolality, pH, A280, RALS, SEC, and DLS. The results of osmolality, pH, A280, and RALS are shown in Tables 109 and 110. [Table 111] [Table 112]

[0239] As shown in Table 109, the concentration of the formulations increased slightly in all formulations after simulated shear stress. As shown in Table 110, the RALS values increased in all formulations after simulated shear stress. Formulations F2 (containing more alanine than arginine) and F3 (containing more arginine than alanine) had very large RALS values, indicating that these formulations are sensitive to shear stress.

[0240] The formulation was subjected to simulated shear stress at 900 RPM for 24 hours, 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 111 and 112. The LMW% was lowest in F1, which contains more alanine than arginine, and F3, which contains more arginine than alanine, in SEC after simulated shear stress. The HMW% increased in all formulations after shear, and F-LF, which contains alanine and trehalose, had the highest main peak%. F-LF, which contains alanine and trehalose, F4, which contains more trehalose than mannitol, and F6, which contains more mannitol than trehalose, had the highest total recovery% after simulated shear stress.

Table 113

Table 114

[0241] The formulation subjected to simulated shear stress was also analyzed by DLS. The results are shown in Table 113. Formulations F-LF, which contains alanine and trehalose, and F4, which contains more trehalose than mannitol, had particle diameters of approximately 1.2 and 1.5 nm for 100% of their volume in DLS, suggesting that these formulations protect SONE-IL12-ABD from shear stress.

Table 115

[0242] In summary, the results of Example 6 demonstrated that arginine protected against heat stress and alanine protected against shear stress. The results further demonstrated that formulations containing a combination of alanine and arginine were more stable against heat stress than against shear stress. Additionally, the results demonstrated that the appearance of the lyophilized cake of Formulation F-LF was relatively improved compared to formulations containing a combination of trehalose and mannitol.

[0243] Example 7: Development of Lyophilization Formulations of SONE-IL12-ABD shown in Table 114 were prepared using a concentration of 20 μg / ml of SONE-IL12-ABD. [Table 116]

[0244] Formulation LF was examined for its thermal properties using modulated differential scanning calorimetry (MDSC). The conditions and results of MDSC are shown in Table 115. [Table 117]

[0245] The formulations shown in Table 114 were also lyophilized according to the lyophilization cycle shown in Table 116. [Table 118]

[0246] The liquid formulations were subjected to temperature ramping RALS and IF to obtain the Tagg and Tm of the formulations. The Tm and Tagg values are shown in Table 117. [Table 119]

[0247] 7.1. Heat Stress Study The liquid and lyophilized formulations were subjected to heat stress at 40 °C for 14 days and monitored for visual appearance, osmolality, pH, A280, RALS, Flowcam, SEC-HPLC, reduced and non-reduced SDS-page, and ELISA. The results of visual appearance are shown in Table 118, osmolality, pH, and RALS in Tables 119 and 120, and A280 in Tables 121 and 122.

[0248] As shown in Table 118, the liquid and reconstituted samples were clear, colorless, and had no visible particles. The lyophilized cakes at T0, T7, and T14 were all white and slightly detached from the glass. Some of the T0 lyophilized cakes were cracked while others were not.

Table 120

Table 121

Table 122

Table 123

Table 124

[0249] The formulations were evaluated for the presence of sub-particle material by Flowcam after incubation at 40 °C for 7 and 14 days. As shown in Table 123, the number of invisible particles increased in the liquid formulation after 14 days of incubation. As shown in Table 124, the number of invisible particles also increased in the lyophilized formulation after 14 days of incubation.

Table 125

Table 126

[0250] The formulation was subjected to heat stress 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 125 to 130. For the liquid formulation at T14, the HMW% increased significantly, the LMW% decreased, and the main peak% decreased. For the lyophilized formulation at T14, the HMW% increased slightly, the main peak% did not change significantly, and the LMW% did not change significantly. For the lyophilized sample at T14, 100% recovery was achieved.

Table 127

Table 128

Table 129

Table 130

Table 131

Table 132

[0251] The liquid and lyophilized formulations LF, which were 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 difference between the liquid and lyophilized formulations.

[0252] The liquid and lyophilized formulations LF were also analyzed by ELISA. The results are shown in Table 131.

Table 133

[0253] 7.2. Freeze-Thaw Studies The formulation LF at T0 was subjected to 5 freeze-thaw cycles and then analyzed for osmolality, pH, RALS, A280, Flowcam, and SEC-HPLC. The results of osmolality, pH, A280, and RALS are shown in Table 132.

Table 134

[0254] The formulation LF was evaluated for the presence of sub-particle substances by Flowcam after 5 freeze-thaw cycles. The results are shown in Table 133. The invisible particles increased after freeze-thaw.

Table 135

[0255] The formulation LF was subjected to 5 freeze-thaw cycles 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 134 and 135. After freeze-thaw, the LMW% was almost halved and the HMW% increased slightly.

Table 136

Table 137

[0256] 7.3. Simulated Shear Stress Study The formulation LF at T0 was subjected to simulated shear stress at 900 RPM at room temperature for 24 hours and analyzed for osmolality, pH, A280, RALS, SEC, and DLS. The results of osmolality, pH, A280, and RALS are shown in Tables 136 and 137.

Table 138

Table 139

[0257] Formulation LF was subjected to simulated shear stress at 900 RPM for 24 hours, 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 138 and 139. By SEC after simulated shear stress, the HMW% increased significantly, the LMW% decreased, and the main peak% decreased. [Table 140] [Table 141]

[0258] Formulation LF subjected to simulated shear stress was also analyzed by DLS. The results are shown in Table 140. [Table 142]

[0259] In summary, the results of Example 7 demonstrated that formulation LF in its lyophilized dosage form was stable at 40°C for 14 days (retaining full activity in ELISA and having no increase in HMW and LMW species).

[0260] Unless otherwise indicated, all numbers representing amounts of ingredients, properties such as molecular weights, reaction conditions, etc. used in the specification and claims are to be understood as being modified in all instances by the term "about". Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and the appended claims are approximations that may vary depending upon the desired properties sought to be obtained by the present disclosure. At a minimum, and not as an attempt to limit the application of the doctrine of equivalents to the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0261] Numerical ranges and parameters setting forth the broad scope of the present disclosure are approximations, although 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 the respective test measurements.

[0262] The terms "a", "an", "the" and similar referents used in the context of describing the present disclosure (especially in the context of the following claims) are to be construed to cover both the singular and the plural forms unless otherwise indicated herein or clearly contradicted by the context. The recitation of a range of values herein is merely intended to serve as a shorthand method of referring individually to each separate value within the range. Unless otherwise indicated herein, each separate value is incorporated herein as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by the context. The use of any examples, or exemplary language (e.g., "such as") provided herein is merely intended to better clarify the present disclosure and does not limit the scope of the present disclosure as otherwise claimed. No language in this specification should be construed as indicating any non-claimed element as essential to the practice of the present disclosure.

[0263] The grouping of alternative elements or embodiments of the present disclosure disclosed herein should not be construed as a limitation. Each group member can be referred to and claimed individually, or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of the group may be included in or deleted from the group for convenience and / or for reasons of patentability. In the event of any such inclusion or deletion, the specification is considered to include the modified group and thus to satisfy the written description of all Markush groups used in the appended claims.

[0264] To implement the present disclosure, certain embodiments of the present disclosure, including the best mode known to the inventor, are described herein. It will be apparent to those skilled in the art that variations of these described embodiments will become apparent upon reading the foregoing description. The inventors expect those skilled in the art to use such variations as appropriate, and the inventors intend for the present disclosure to be practiced otherwise than as specifically described herein. Accordingly, the present disclosure includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Further, unless otherwise indicated herein or clearly contradicted by context, any combination of the above-described elements in all possible variations is included in the present disclosure.

[0265] The specific embodiments disclosed herein may be further limited in the claims using the language “consisting of” or “consisting essentially of”. When used in the claims, whether applied or added by amendment, the transitional term “consisting of” excludes any element, step, or ingredient not specified in the claims. The transitional term “consisting essentially of” limits the scope of the claim to the specified material or step and those that do not materially affect the basic or novel characteristics. The embodiments of the present disclosure so claimed are possible as essentially or explicitly described herein.

[0266] It should be understood that the embodiments of the present disclosure disclosed herein are illustrative of the principles of the present disclosure. Other modifications that can be used are within the scope of the present disclosure. Accordingly, by way of example and not limitation, alternative configurations of the present disclosure can be utilized in accordance with the teachings herein. Accordingly, the present disclosure is not limited to that which is precisely shown and described.

[0267] Although the present disclosure has been described and illustrated herein by reference to various specific materials, procedures, and examples, it is to be understood that the disclosure is not limited to the particular combinations of materials and procedures selected for that purpose. As will be understood by those skilled in the art, numerous variations of such details may be shown. The specification and examples are to be regarded as merely illustrative, and the true scope and spirit of the disclosure are intended to be indicated by the following claims. All references, patents, and patent applications cited in this application are hereby incorporated by reference in their entirety.

Claims

1. - Fusion protein of Sequence ID No. 11 with a concentration of approximately 10 μg / mL to approximately 50 μg / mL, - Alanine in concentrations of approximately 25 mM to 100 mM, - Trehalose in a concentration of approximately 100 mM to 400 mM, - Glycylglycine at approximately 10 mM to 50 mM, - Approximately 0.01% to approximately 0.04% polysorbate 20 (v / v), and - A stable liquid pharmaceutical preparation containing approximately 5 μM to approximately 20 μM of diethylenetriaminepentaacetic acid (DTPA), The liquid pharmaceutical preparation having a pH of approximately 6.8 to approximately 8.

0.

2. The aforementioned formulations are approximately 10 μg / mL to approximately 30 μg / mL, approximately 15 μg / mL to approximately 25 μg / mL, approximately 10 μg / mL to approximately 15 μg / mL, approximately 15 μg / mL to approximately 20 μg / mL, approximately 20 μg / mL to approximately 25 μg / mL, approximately 25 μg / mL to approximately 30 μg / mL, approximately 30 μg / mL to approximately 35 μg / mL, approximately 35 μg / mL to approximately 40 μg / mL, approximately 40 μg / mL to approximately 45 μg / mL, and approximately 45 μg / mL to approximately 50 μg / mL. The liquid formulation according to claim 1, comprising the fusion protein of SEQ ID NO: 11 in an amount of 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, preferably the formulation comprising 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 according to claim 1, wherein the formulation contains approximately 20 μg / mL of the fusion protein of Sequence ID No.

11.

4. The aforementioned formulation contains alanine in concentrations of approximately 25 mM to 75 mM, 40 mM to 60 mM, 25 mM to 30 mM, 30 mM to 40 mM, 40 mM to 50 mM, 50 mM to 60 mM, 60 mM to 70 mM, 70 mM to 80 mM, 80 mM to 90 mM, and 90 mM to approximately A liquid formulation according to any one of claims 1 to 3, comprising 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 the formulation comprising about 50 mM alanine.

5. The aforementioned formulation contains approximately 100 mM to approximately 300 mM trehalose, approximately 150 mM to approximately 250 mM trehalose, approximately 100 mM to approximately 150 mM trehalose, approximately 150 mM to approximately 200 mM trehalose, approximately 200 mM to approximately 250 mM trehalose, approximately 250 mM to approximately 300 mM trehalose, approximately 300 mM to approximately 350 mM trehalose, and approximately 350 mM to approximately 400 mM trehalose. A liquid formulation according to any one of claims 1 to 3, comprising M 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 the formulation comprising about 200 mM trehalose.

6. The aforementioned formulation contains approximately 10 mM to approximately 40 mM glycylglycine, approximately 10 mM to approximately 30 mM glycylglycine, approximately 10 mM to approximately 15 mM glycylglycine, approximately 15 mM to approximately 20 mM glycylglycine, approximately 20 mM to approximately 25 mM glycylglycine, approximately 25 mM to approximately 30 mM glycylglycine, approximately 35 mM to approximately 40 mM glycylglycine, approximately 40 mM to approximately 45 mM glycylglycine, and approximately 45 mM to approximately 50 mM glycylglycine. A liquid formulation according to any one of claims 1 to 3, comprising approximately 10 mM glycylglycine, approximately 15 mM glycylglycine, approximately 20 mM glycylglycine, approximately 25 mM glycylglycine, approximately 30 mM glycylglycine, approximately 35 mM glycylglycine, approximately 40 mM glycylglycine, approximately 45 mM glycylglycine, and / or approximately 50 mM glycylglycine, preferably the formulation comprising approximately 25 mM glycylglycine.

7. The liquid formulation according to any one of claims 1 to 3, 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 the formulation comprises about 0.02% polysorbate 20 (v / v).

8. The liquid formulation according to any one of claims 1 to 3, wherein the formulation comprises about 5 μM to about 15 μM of DTPA, about 5 μM to about 10 μM of DTPA, about 10 μM to about 15 μM of DTPA, about 15 μM to about 20 μM of DTPA, about 5 μM of DTPA, about 10 μM of DTPA, about 15 μM of DTPA, and / or about 20 μM of DTPA, preferably the formulation comprises about 10 μM of DTPA.

9. The liquid formulation according to any one of claims 1 to 3, wherein the formulation has a pH of approximately 7.0 to approximately 7.8, approximately 7.2 to approximately 7.8, approximately 7.4 to approximately 7.6, approximately 7.0 to approximately 7.2, approximately 7.2 to approximately 7.4, approximately 7.4 to approximately 7.6, approximately 7.6 to approximately 7.8, approximately 7.8 to approximately 8.0, approximately 6.8, approximately 6.9, approximately 7.0, approximately 7.1, approximately 7.2, approximately 7.3, approximately 7.4, approximately 7.5, approximately 7.6, approximately 7.7, approximately 7.8, approximately 7.9, and / or approximately 8.0, preferably the formulation has a pH of approximately 7.

5.

10. - Approximately 20 μg / mL of the fusion protein of Sequence ID No. 11, - Approximately 50 mM alanine, - Approximately 200 mM trehalose, - Approximately 25 mM glycylglycine, - Approximately 0.02% polysorbate 20 (v / v), and - A liquid pharmaceutical formulation containing approximately 10 μM of DTPA, The liquid pharmaceutical preparation having a pH of approximately 7.

5.

11. The liquid formulation according to any one of claims 1 to 3 and 10, wherein the formulation is a reconstituted formulation.

12. The liquid formulation according to claim 11, wherein the reconstituted formulation is reconstituted from a lyophilized formulation.

13. - Approximately 0.01% to approximately 0.03% (w / w) of the fusion protein of Sequence ID No. 11, - Approximately 2% to 8% alanine (w / w), - Approximately 50% to 95% trehalose (w / w), - Approximately 2% to 8% glycylglycine (w / w), - Approximately 0.1% to approximately 0.4% polysorbate 20 (w / w), and - A solid pharmaceutical formulation containing approximately 0.002% to approximately 0.006% diethylenetriaminepentaacetic acid (DTPA) (w / w).

14. - Approximately 0.024 to approximately 0.028% (w / w) of the fusion protein of Sequence ID No. 11, - Approximately 4.0% and 5.0% alanine (w / w), - Approximately 70% to 80% trehalose (w / w), - Approximately 3.0% to 4.0% glycylglycine (w / w), - Approximately 0.15% to approximately 0.30% polysorbate 20 (w / w), and - A solid pharmaceutical formulation according to claim 13, comprising approximately 0.0045% to approximately 0.0055% DTPA (w / w).

15. The formulation according to claim 13 or 14, wherein the formulation is a freeze-dried formulation.

16. The formulation according to any one of claims 1 to 3, 10, and 13, wherein the formulation is suitable for parenteral administration.

17. The preparation according to any one of claims 1 to 3, 10, and 13, wherein the preparation is suitable for intravenous administration.

18. The formulation according to any one of claims 1 to 3, 10, and 13, wherein the formulation is suitable for subcutaneous administration.

19. A formulation according to any one of claims 1 to 3, 10, and 13, for use in a method of treating IL-12-related disease or disorder in a subject requiring treatment for IL-12-related disease or disorder.

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