Method for producing recombinant IL-12 albumin-binding domain fusion proteins

Abstract

This specification provides a method for producing recombinant IL-12 albumin-binding domain (ABD) fusion proteins. The method in question advantageously includes a series of downstream purification steps that lead to high-yield production of purified cytokine-based proteins.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 490,202, filed on 14 March 2023, which is incorporated herein by reference in its entirety. [Background technology]

[0002] IL-12 is known as a T cell stimulator that can stimulate the proliferation 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 cytotoxic activity in NK cells and CD8+ cytotoxic T lymphocytes. Furthermore, IL-12 can also possess anti-angiogenic properties by increasing the production of interferon-gamma, which in turn increases the production of chemokine-inducible protein-10 (IP-10 or CXCL10). While not bound by any specific theory of action, IL-12 is thought to be usable to treat cancer through its ability to induce immune responses and its anti-angiogenic properties.

[0003] A short circulating half-life is a major obstacle for many biologics, including cytokine-based therapies. See, for example, Perdreau et al., European Cytokine Network 21:297-307 (2010). These short-acting therapies require frequent dosing profiles, which can reduce their clinic applicability, particularly for chronic conditions. Specifically, cytokines have been shown to be highly toxic when administered repeatedly. See, for example, van der Poll et al., Cytokines as Regulators of Coagulation, Madame Curie Bioscience Database 2000. A long serum half-life is desirable because it reduces the need for frequent injections of the molecule to achieve therapeutically relevant serum concentrations and doses low enough to be patient-tolerated.

[0004] The cyclic half-life of cytokines can be extended by conjugating them to an albumin-binding domain (ABD). Albumin-binding domain (ABD) fusion proteins have been shown to be useful in extending the half-lives of biologics (e.g., interleukins and antibodies). Serum albumin has a long half-life ranging from 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 within the acidic environment of early endosomes. Albumin-FcRn binding bypasses albumin molecules from degradation in the lysosomal compartment and reorients them to the plasma membrane, where they are released to return to the plasma due to the neutral pH. The albumin-binding domain (ABD) does not compete with FcRn for albumin binding and binds to albumin within a pH range that allows ABD to undergo FcRn-driven endosomal albumin recycling once bound to albumin. Thus, cytokine fusion proteins containing such albumin-binding domains (ABDs) have the ability to evade lysosomal degradation using the albumin-FcRn pathway, and consequently exhibit a longer serum half-life than their ABD-less counterparts.

[0005] While cytokine-based biologics such as IL-12 have therapeutic potential, challenges remain in removing proteolytic enzymes and protein aggregation during the manufacturing of these biologics. Interleukins are known to secrete proteolytic enzymes and cause aggregation during manufacturing. The effects of proteolysis are that cell culture contaminants and proteolytic enzymes are spontaneously secreted into the culture medium by cytokines, causing clipping or degradation of intact molecules. Therefore, there remains a need for improved methods for manufacturing cytokine-based molecules, including IL-12-based therapies. [Overview of the project]

[0006] This specification provides a method for producing an IL-12 albumin-binding domain fusion protein. The method in question advantageously includes a series of downstream purification steps that lead to high-yield production of purified IL-12 albumin-binding domain fusion protein.

[0007] In one embodiment, a method for producing an IL-12 albumin-binding domain (ABD) fusion protein is provided herein, comprising: a) culturing a plurality of mammalian host cells, each containing a polynucleotide encoding a protein, in a bioreactor containing a liquid culture medium, wherein the host cells are cultured under conditions in which the protein is produced and secreted into the liquid culture medium by the host cells; b) removing the liquid culture medium containing the secreted protein from the bioreactor; c) purifying the protein by passing the liquid culture medium removed from the bioreactor directly through one or more chromatography columns, wherein one or more chromatography columns are operably and continuously linked to a perfusion bioreactor; and recovering the purified protein from the liquid culture medium.

[0008] In some embodiments, the bioreactor is a perfusion-mode bioreactor. In some embodiments, the liquid culture medium containing the secreted protein is removed from the perfusion-mode bioreactor and replaced with fresh liquid culture medium at set time intervals. In exemplary embodiments, the set time intervals are 12 hours, 24 hours, 48 ​​hours, or 72 hours.

[0009] In some embodiments, the liquid culture medium containing the secreted protein is continuously removed from the perfusion-mode bioreactor and continuously replaced with fresh liquid culture medium.

[0010] In some embodiments, the liquid culture medium is removed from the bioreactor at least about 96 hours after the start of culture step a). In certain embodiments, the liquid culture medium containing the secreted protein is removed from the perfusion-mode bioreactor and replaced with an equal volume of fresh liquid culture medium. In some embodiments, a portion of the volume of the liquid culture medium containing the secreted protein is partially removed from the perfusion-mode bioreactor and replaced with an equal volume of fresh liquid culture medium.

[0011] In certain embodiments, host cells are prevented from leaving the perfusion bioreactor during removal step b) using a cell separation system.

[0012] In some embodiments, host cells are cultured in a liquid culture medium in the presence of nutrients that are periodically replenished.

[0013] In some embodiments, multiple mammalian host cells are grown in a bioreactor at a rate of 20-100 × 10⁶ 6 The cells are cultured in a liquid culture medium at a density of cells / mL.

[0014] In some embodiments, the purification step c) separates one or more proteolytic enzymes from the protein.

[0015] In some embodiments, the albumin-binding domain of the IL-12 ABD fusion protein includes a variable heavy chain domain or a variant thereof containing SEQ ID NO: 1, and a variable light chain domain or a variant thereof containing SEQ ID NO: 5. In a particular embodiment, the albumin-binding domain is an scFv containing the amino acid sequence or a variant thereof of SEQ ID NO: 9.

[0016] In some embodiments, the IL-12 in the IL-12 fusion protein is single-stranded IL-12 containing a p35 subunit covalently bonded to a p40 subunit. In exemplary embodiments, the p40 subunit has the amino acid sequence of SEQ ID NO: 10 or a variant thereof. In some embodiments, the p35 subunit has the amino acid sequence of SEQ ID NO: 11 or a variant thereof. In some embodiments, the single-stranded IL-12 has the amino acid sequence of SEQ ID NO: 12. In some embodiments, the IL-12 ABD fusion protein has the amino acid sequence of SEQ ID NO: 13.

[0017] In some embodiments, one or more chromatography columns include ion exchange chromatography columns, hydrophobic interaction columns, affinity columns, pseudoaffinity columns, or size exclusion chromatography columns. In exemplary embodiments, one or more chromatography columns include affinity columns. In some embodiments, the pseudoaffinity column includes a cibacron blue ligand. In exemplary embodiments, the pseudoaffinity column includes an affinity chromatography medium as shown in Figure 14. [Brief explanation of the drawing]

[0018] [Figure 1] Figure 1 provides a schematic diagram of an exemplary embodiment of a method for producing the subject provided herein. Host cells are grown in a standard stirred-tank bioreactor. Perfusion is achieved by a continuous supply of fresh culture medium. An ATF device continuously removes the IL-12 albumin-binding domain fusion protein product along with the used medium. The perfusion fluid is passed through a capture column operated in parallel mode, with the recovered product alternating between two columns (columns 1 and 2), to achieve continuous capture of the IL-12 albumin-binding domain fusion protein product. The product can be eluted from the column, for example, by using conduction such as high conductivity or pH, which leads to the dissociation of the bound product. [Figure 2]Figure 2 illustrates a schematic diagram of an exemplary embodiment of an upstream process of a manufacturing method of the subject matter provided herein. In such embodiments, cells (e.g., CHO cells) are first grown in shake flasks and then inoculated into a perfusion bioreactor at an inoculation viable cell density range of 1.2 - 1.8×106 cells / mL and a target seeding density of 1.5×106 cells / mL. In some embodiments, the perfusion rate is started at 0.5RV and increases as the culture time and VCD increase. In some embodiments, the bioreactor uses an alternating tangential flow (ATF) filter to enable the recovery of the target recombinant protein (e.g., IL-12 albumin binding domain fusion protein) secreted into the culture medium. In some embodiments, the recovered protein is purified using an in-line chromatography system comprising one or more different chromatography columns. [Figure 3] Figure 3 illustrates a schematic diagram of an exemplary embodiment of a downstream process of a manufacturing method of the subject matter provided herein. In such embodiments, the culture medium containing the target protein (e.g., IL-12 albumin binding domain fusion protein) recovered from a continuous perfusion process is fed to a chromatography system for isolation and purification of the target protein. In some embodiments, the target protein undergoes two or more chromatography processes. In an exemplary embodiment, the target protein is subjected to affinity chromatography. In further embodiments, the target protein is further subjected to hydrophobic interaction (HIC) chromatography and / or cation exchange (CEX) chromatography. After chromatographic purification, the target protein may further undergo a diafiltration / ultrafiltration and / or nanofiltration step. [Figure 4] Figure 4 illustrates the albumin binding domain component of an IL-12 albumin binding domain fusion protein that can be manufactured using the method of the subject matter provided herein. [Figure 5]Figure 5 illustrates an exemplary IL-12 albumin binding domain (ABD) fusion protein that can be produced using the methods of the subject matter provided herein. The illustrated IL-12 is a single-chain IL-12 molecule in which the IL-12A (p35) and IL-12B (p40) components are joined using a polypeptide linker. [Figure 6] Figure 6 illustrates the evaluation of viable cell density and cell viability (A) and residual glucose (B) during the production of an IL-12 ABD fusion protein using the continuous perfusion culture method (20L) of the subject matter described herein. Parameters were evaluated from aliquots of the culture medium taken every 24 hours. [Figure 7] Figure 7 illustrates the evaluation of the lactate profile (A) and glutamine profile (B) during the production of an IL-12 ABD fusion protein using the continuous perfusion culture method (20L) of the subject matter described herein. Parameters were evaluated from aliquots of the culture medium taken every 24 hours. [Figure 8] Figure 8 illustrates the evaluation of the ammonia profile (A) and glutamine profile (B) during the production of an IL-12 ABD fusion protein using the continuous perfusion culture method (20L) of the subject matter described herein. Parameters were evaluated from aliquots of the culture medium taken every 24 hours. [Figure 9] Figure 9 provides an exemplary operating process flow for the scale-up production of an IL-12 ABD fusion protein using the methods of the subject matter provided herein. [Figure 10] Figure 10 illustrates the affinity chromatography profile for a cycle of scale-up production of an IL-12 / ABD fusion protein using the methods of the subject matter provided herein. The affinity column contains Capto(™) Blue medium for albumin capture. [Figure 11]Figure 11 illustrates the average loading density (mg / mL resin) and recovery of the protein in question during an exemplary chromatographic purification step in the production of an IL-12 / ABD fusion protein using the subject method provided herein. [Figure 12] Figure 12 illustrates a typical reverse-phase high-performance liquid chromatography (RP-HPLC) profile of the Capto® Blue affinity chromatography purification step during the production of IL-12 / ABD fusion protein using the subjective method provided herein. [Figure 13] Figure 13 illustrates a typical reverse-phase high-performance liquid chromatography (RP-HPLC) profile of the Capto® Blue affinity chromatography purification step during the production of IL-12 / IL-15 ABD fusion protein (SON1210) using the subjective method provided herein. [Figure 14] Figure 14 illustrates exemplary pseudo-affinity ligands that contain cibacron blue and can be used for the purification of IL-12 / ABD fusion proteins as described herein. [Modes for carrying out the invention]

[0019] I. Overview This specification provides a method for producing an IL-12 albumin-binding domain fusion protein. In embodiments of the method provided herein, the IL-12 albumin-binding domain fusion protein is produced in a bioreactor connected immediately downstream of a purification system for purification. In several embodiments, the purification system includes one or more chromatographic columns for purification. In several embodiments, after the protein is produced and secreted into a liquid culture medium in the bioreactor, the liquid culture medium containing the protein passes directly to the purification system (e.g., one or more columns for purification), and the resulting purified product is recovered. When the purification system (e.g., one or more chromatographic columns) is directly and operably connected to the bioreactor, the protein is passed in a continuous manner from the bioreactor to the purification process, thereby minimizing contact time with proteolytic enzymes secreted during the production process that can cause clipping and degradation. In contrast, previous cytokine production methods often include a retention step in which the protein is held for a certain period before purification, which increases the exposure time to such proteolytic enzymes and leads to proteolysis. Thus, the method of the subject matter advantageously enables the high-yield production of IL-12 albumin-binding domain fusion proteins that exhibit less degradation than those produced using previous methods. Aspects of the method of the subject matter are described in more detail.

[0020] II. Definition To ensure that this application can be fully understood, some definitions are provided below. These definitions are intended to encompass grammatical equivalents.

[0021] The terms “a” or “an” can refer to one or more of its entities, i.e., to multiple references. Therefore, the terms “a” or “an,” “one or more,” and “at least one” are used interchangeably herein. In addition, a reference to “one element” with the indefinite article “a” or “an” does not rule out the possibility of two or more elements existing unless the context explicitly requires that only one or one of the elements exists.

[0022] It should be noted that claims may be drafted to exclude any optional elements. Thus, this statement is intended to serve as an antecedent to the use of such exclusive terms, such as “solely,” “only,” and similar terms, or the use of “negative” restrictions, in relation to the enumeration of elements of the claims. As will be apparent to those skilled in the art by reading this disclosure, each of the individual embodiments described and illustrated herein has distinct components and features that are readily separable from or combined with any of the features of several other embodiments without departing from the scope or spirit of the invention. Any of the enumerated methods may be performed in the order of the enumerated events, or in any other logically possible order. Any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the invention, but representative illustrative methods and materials are described herein.

[0023] The ranges provided herein are understood to be all abbreviated representations of the values ​​within that range. For example, the range 1 to 50 is understood to include any number, combination of numbers, or subrange from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, and all decimal values ​​that lie between the aforementioned integers, such as 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9. With respect to subranges, the specific intention is to have "nested subranges" extending from either endpoint of the range. For example, nested subranges of the exemplary range 1-50 may include 1-10, 1-20, 1-30, and 1-40 in one direction, and 50-40, 50-30, 50-20, and 50-10 in the other direction.

[0024] Unless otherwise specified or evident from the context, the term “about” as used herein is understood to mean within the normal tolerances in the art, for example, within two standard deviations of the mean. “About” can be understood to mean within 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise evident from the context, all numerical values ​​provided herein are modified by the term “about.”

[0025] Before further describing the present invention, it should be understood that the invention is not limited to the specific embodiments described and may, of course, be modified in this way. Furthermore, it should be understood that the scope of the invention is limited only by the appended claims, and therefore the terms used herein are for the sole purpose of describing specific embodiments and are not intended to be limiting.

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art in which the invention pertains. Where a range of values ​​is provided, unless the context otherwise explicitly indicates, each intermediate value between the upper and lower limits of that range, up to one-tenth of the lower limit, and any other stated or intermediate values ​​within that stated range, are understood to be included in the invention. The upper and lower limits of these smaller ranges may independently be included in smaller ranges, which are included in the invention and subject to any specifically excluded limitations within the stated range. Where a stated range includes one or both of the limitations, a range that excludes either or both of those included limitations is also included in the invention. A particular range is presented herein with a numerical value preceded by the term “approximately”. In this specification, the term “approximately” is used to provide a letter suffix to a number that is close to or approximates the number following the term, as well as to the exact number that follows it. When determining whether a number is close to or approximates a specifically listed number, a close or approximate, unlisted number may be substantially equivalent to the specifically listed number in the given context.

[0027] All publications, patents, and patent applications cited herein are incorporated by reference as if each individual publication, patent, or patent application were specifically and individually indicated as being incorporated by reference. Furthermore, each cited publication, patent, or patent application is incorporated by reference to disclose and describe the subject matter that the publication is related to. Any citation of a publication is for disclosure prior to the filing date and should not be construed as acknowledging that the invention described herein is not prior to such publication for the sake of prior art. Furthermore, the dates of publications provided may differ from the actual publication dates and may need to be verified independently.

[0028] III. IL-12 albumin-binding domain fusion protein In several embodiments, the subjective methods described herein are useful for producing IL-12 albumin-binding domain (ABD) fusion proteins. Such IL-12 ABD fusion proteins are used, for example, in the treatment of cancer. In some embodiments, the IL-12 albumin-binding domain fusion protein includes an albumin-binding domain comprising an antibody variable heavy chain domain having vhCDR1 having the amino acid sequence of SEQ ID NO: 2, vhCDR2 having the amino acid sequence of SEQ ID NO: 3, and vhCDR3 having the amino acid sequence of SEQ ID NO: 4 (see Figure 4). In some embodiments, the albumin-binding domain includes a variable light chain domain having vlCDR1 having the amino acid sequence of SEQ ID NO: 6, vlCDR2 having the amino acid sequence of SEQ ID NO: 7, and vlCDR3 having the amino acid sequence of SEQ ID NO: 8. In several embodiments, the albumin-binding domain includes a variable heavy chain having the amino acid sequence of SEQ ID NO: 1, and / or a variable light chain having the amino acid sequence of SEQ ID NO: 5. In several embodiments, the albumin-binding domain includes a variable heavy chain having the amino acid sequence of SEQ ID NO: 1, and a variable light chain having the amino acid sequence of SEQ ID NO: 5. In several embodiments, the albumin-binding domain of the IL-12 albumin-binding domain fusion protein is an scFv having the amino acid sequence A10m3 (SEQ ID NO: 9).

[0029] In several embodiments, the IL-12 albumin-binding domain fusion protein includes an albumin-binding domain that is a variant of the A10m3 albumin-binding domain as shown in Figure 4. In exemplary embodiments, the albumin-binding domain of the IL-12 albumin-binding domain fusion protein includes a set of six CDRs having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications, as shown in Figure 4, compared to the six CDRs of A10m3. In several embodiments, the albumin-binding domain of the IL-12 albumin-binding domain fusion protein includes six CDRs that are at least 90%, 95%, 97%, 98%, or 99% identical to the six CDRs of A10m3 (see Figure 4). In several embodiments, the albumin-binding domain includes a VH domain and / or VL domain having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid changes from the VH domain and / or VL domain of A10m3, as illustrated in Figure 4. In several embodiments, the albumin-binding domain includes a VH domain and / or VL domain that are at least 90%, 95%, 97%, 98%, or 99% identical to the VH and / or VL domain of A10m3, as illustrated in Figure 4. In certain embodiments, variants of the A10m3 albumin-binding domain have the ability to bind human serum albumin, as measured by at least one of the following assays: Biacore assay, surface plasmon resonance (SPR) assay, flow cytometry assay, and / or BLI (biolayer interferometry, e.g., Octet assay), the latter of which is particularly used in many embodiments.

[0030] In certain embodiments, IL-12 is a single-stranded IL-12 polypeptide comprising an IL-12 p35 subunit linked to an IL-12 p40 subunit. In several embodiments, the IL-12 single-stranded polypeptide advantageously retains one or more of the biological activities of wild-type IL-12. In some embodiments, the single-stranded IL-12 polypeptide described herein follows 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-stranded IL-12 follows the formula (p35)-(L)-(p40) from the N-terminus to the C-terminus. Any suitable linker can be used with the single-stranded IL-12 polypeptide. A preferred linker is, for example, the amino acid sequence (GGGGS). x Examples of linkers include those having the formula, where x is an integer from 1 to 10. Other suitable linkers include, for example, the amino acid sequence GGGGGS. Exemplary single-stranded IL-12 linkers that can be used with the single-stranded IL-12 polypeptide of the subject are also described in Lieschke et al., Nature Biotechnology 15:35-40 (1997), which is incorporated herein by reference and in particular for its teaching of the IL-12 polypeptide linker.

[0031] In some embodiments, the IL-12 albumin-binding domain fusion protein IL-12 includes a human p40 subunit having the amino acid sequence of SEQ ID NO: 10. In some embodiments, IL-12 includes a variant human p40 subunit having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid changes compared to SEQ ID NO: 10. In some embodiments, IL-12 includes a variant human p40 that is at least 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 10.

[0032] In some embodiments, IL-12 in the IL-12 albumin-binding domain fusion protein includes a human p35 subunit having the amino acid sequence of SEQ ID NO: 11. In some embodiments, IL-12 includes a variant human p35 subunit having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid changes compared to SEQ ID NO: 10. In some embodiments, IL-12 includes a variant human p40 that is at least 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 11.

[0033] IV. Cell culture In some embodiments of the subject method, the IL-12 albumin-binding domain fusion protein is produced in a bioreactor by culturing mammalian host cells containing the polynucleotide encoding the protein within the bioreactor. In some embodiments, the host cells are cultured in a liquid culture medium under conditions in which the protein is produced and secreted by the host cells into the liquid culture medium.

[0034] Any suitable host cell can be used for the production of the IL-12 albumin-binding domain fusion protein. In exemplary embodiments, the host cell is a mammalian host cell. A wide variety of mammalian cell lines suitable for growth in culture are available from the American Type Culture Collection (Manassas, Virginia) and commercial vendors. Examples of cells that can be used in the method of the subject include, but are not limited to, VERO, BHK, HeLa, CV1 (including Cos), MDCK, 293, 3T3, myeloma cell lines (e.g., NSO, NSL), PC12, WI38 cells, and Chinese hamster ovary (CHO) cells. CHO cells are widely used for the production of recombinant complex proteins, such as cytokines, coagulation factors, and antibodies (Brasel et al. (1996), Blood 88:2004-2012; Kaufman et al. (1988), J. Biol Chem 263:6352-6362; McKinnon et al. (1991), J Mol Endocrinol 6:231-239; Wood et al. (1990), J. Immunol. 145:3011-3016). Dihydrofolate reductase (DHFR)-deficient mutant cell lines (Urlaub et al. (1980), Proc Natl Acad Sci USA 77:4216-4220), DXB11 and DG-44, and CHO-K1 are desirable CHO host cell lines because efficient DHFR-selectable and amplified gene expression systems enable high levels of recombinant protein expression in these cells (Kaufman RJ. (1990), Meth Enzvmol 185:537-566). In addition, these cells are easy to handle as adherent or suspension cultures and exhibit relatively good genetic stability. CHO cells and the proteins recombinantly expressed within them are extensively characterized and approved by regulatory authorities for use in clinical and commercial manufacturing.

[0035] Host cells expressing recombinant IL-12 albumin-binding domain fusion protein can be cultured in any suitable medium that allows for host cell proliferation and expression of the recombinant protein. Cell culture medium formulations are well known in the art. Typically, cell culture media contain buffers, salts, carbohydrates, amino acids, vitamins, and trace essential elements. Cell culture media may or may not contain serum, peptones, and / or proteins. A variety of cell culture media are commercially available, including serum-free and defined culture media. For example, any one or any combination of the following cell culture media can be used: RPMI-1640 medium, RPMI-1641 medium, Dulbecco's modified Eagle medium (DMEM), Sartorius Stedim Cellca smd (CHOKO), Cellca FMA, Cellca FMB (SAFC), Acti-Pro (GE), Minimum Essential Medium Eagle, F-12K medium, Ham F12 medium, Iskov modified Dulbecco medium, McCoy's 5A medium, Leibowitz L-15 medium, and serum-free media such as EX-CELL® 300 series (JRH Biosciences, Lenexa, Kansas). Cell culture media may be supplemented with additional or increased concentrations of components such as amino acids, salts, sugars, vitamins, hormones, growth factors, buffers, antibiotics, lipids, trace elements, and the like, depending on the requirements of the cells being cultured and / or the desired cell culture parameters. Cell culture media may be serum-free, protein-free, and / or peptone-free. “Serum-free” applies to cell culture media that do not contain animal serum, such as fetal bovine serum. “Protein-free” applies to cell culture media that do not contain exogenously added proteins, such as transferrin, protein growth factor IGF-1, or insulin. Protein-free media may or may not contain peptone. “Peptone-free” applies to cell culture media that do not contain exogenous protein hydrolysates, such as animal and / or plant protein hydrolysates. Removing serum and / or hydrolysates from cell culture media has the advantage of reducing lot-to-lot variability and enhancing processing steps such as filtration.However, when serum and / or peptones are removed from the cell culture medium, cell proliferation, viability, and / or protein expression may be reduced or fall below optimal levels. Therefore, serum-free and / or peptone-free cell culture media may be highly enriched with amino acids, trace elements, and similar substances. See, for example, U.S. Patents 5,122,469 and 5,633,162. A defined cell culture medium formulation is a complex containing amino acids, inorganic salts, carbohydrates, lipids, vitamins, buffers, and trace essential elements. The necessary and beneficial components for maintaining a cell culture with desired properties depend on the specific host cells used and the way the bioreactor is operated.

[0036] Host cells may be cultured using any preferred technique that allows for cell proliferation and expression of the IL-12 albumin-binding domain fusion protein. Mammalian cells may be cultured in suspension or attached to a solid substrate. In some embodiments, mammalian cells are cultured in a bioreactor. Exemplary bioreactors include, but are not limited to, fluidized bed bioreactors, hollow fiber bioreactors, roller bottles, shaking flasks, or agitated tank bioreactors, with or without microcarriers. The bioreactor can operate in batch, fed-batch, continuous, semi-continuous, or perfusion mode. In certain embodiments, the culture is a large-scale culture carried out in a volume of culture medium of at least about 10 L, at least about 20 L, at least about 25 L, at least about 50 L, at least about 75 L, at least about 100 L, at least about 500 L, at least about 1000 L, at least about 1000 L, at least about 2000 L, at least about 3000 L, at least about 5000 L, at least about 7000 L, at least about 8000 L, at least about 10000 L, at least about 15000 L, or at least about 20000 L. In some embodiments, the culture process is carried out in a volume of culture medium of 30 mL to 50 L. In several embodiments, the culture process is carried out in a volume of culture medium of 1 L to 10 L, 10 L to 20 L, 20 L to 50 L, or 50 L to 100 L.

[0037] In several embodiments, mammalian cells are cultured in a fed-batch mode. A fed-batch mode refers to a culture of mammalian cells that is supplied with a concentrated feed medium containing nutrients, either continuously or periodically. In several embodiments, the supply occurs on a predetermined schedule, for example, daily, every two days, or every three days. The culture can be monitored for tyrosine, cystine, and / or cysteine ​​levels in the culture medium and can be adjusted by supplying concentrated tyrosine solution or tyrosine and cysteine ​​solution to maintain tyrosine, cysteine, and / or cystine within a desired range. Compared to batch culture where no supply occurs, fed-batch culture can produce larger quantities of protein. In several embodiments, mammalian cells are cultured in a continuous fed-batch mode. In several embodiments, mammalian cells are cultured in a periodic fed-batch mode.

[0038] In exemplary embodiments, the bioreactor operates in perfusion mode. In perfusion mode, the IL-12 albumin-binding domain fusion protein is removed from the bioreactor at regular intervals and replaced with fresh medium. Thus, the host cells are not exposed to increasing concentrations of toxic byproducts (e.g., proteolytic enzymes) generated during the cell culture process, thereby minimizing degradation. In some embodiments, the IL-12 albumin-binding domain fusion protein is continuously removed along with the medium and replaced with an equal volume of fresh medium. In some specific embodiments, the IL-12 albumin-binding domain fusion protein is removed along with the medium at fixed, regular time intervals and replaced with an equal volume of fresh medium. In some embodiments, the time interval is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours. In some embodiments, the time interval is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days, or more.

[0039] In embodiments under perfusion conditions, cell culture can proceed for many weeks to many months. In some embodiments, the bioreactor is operated under perfusion conditions for about 1, 2, 3, 4, 5, 6, or 7 days. In certain embodiments, the bioreactor is operated under perfusion conditions for 1, 2, 3, or 4 weeks. In some embodiments, the bioreactor is operated under perfusion conditions for up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 12 months.

[0040] In several embodiments, cell culture is maintained at approximately 20°C to 50°C. In several embodiments, the reaction is carried out at approximately 25°C to 45°C. In some embodiments, the reaction is carried out at approximately 30°C to 40°C. In some embodiments, the cell culture temperature is maintained at the same temperature while the cells are in the proliferation and production phases. In several embodiments, the cell culture temperature is within approximately 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, or 10°C during the proliferation and production phases.

[0041] In several embodiments, the pH of the cell culture is maintained at approximately 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0. In several embodiments, the pH of the cell culture is maintained between approximately 6.0 and approximately 8.0. In some embodiments, the pH of the cell culture is maintained between approximately 6.5 and 7.5.

[0042] In exemplary embodiments, the bioreactor operates under perfusion conditions that allow host cells to grow at high density. In specific embodiments, the host cells are at least 10 × 10 6 cells / mL, 15×10 6 cells / mL, 25×10 6 cells / mL, 30×10 6 cells / mL, 35×10 6 cells / mL, 40×10 6 cells / mL, 45×106 cells / mL, 50×10 6 cells / mL, 55×10 6 cells / mL, 60×10 6 cells / mL, 70×10 6 cells / mL, 80×10 6 cells / mL, 90×10 6 cells / mL, or 100×10 6 cells / mL and grown to a density of. Growing the cells at high density allows for the production of more IL-12 albumin binding domain fusion protein in the same volume of medium over a standard perfusion process.

[0043] In multiple embodiments, the host cells are first, within a bioreactor, at about 0.5×10 6 cells / mL to about 3.0×10 6 cells / mL and seeded at a cell density of. In multiple embodiments, the host cells are first, within a bioreactor, at about 1×10 6 cells / mL to about 2.0×10 6 cells / mL and seeded at a cell density of. In certain embodiments, the host cells are capable of passing through multiple growth phases to maximize protein production. In an exemplary embodiment, the host cells pass through at least 2, 3, 4, 5, 6, 7, 8, 9, 10 growth phases within the bioreactor prior to the final production phase.

[0044] V. Purification In embodiments of the subject method, the culture medium containing the IL-12 albumin-binding domain fusion protein produced during the cell culture step is removed from the bioreactor and purified by directly supplying the culture medium containing the IL-12 albumin-binding domain fusion protein to a purification system. The resulting purified product is recovered. In some embodiments, the purification system includes one or more chromatography columns. The purification system (e.g., one or more chromatography columns) is directly and operably connected to the bioreactor so that the IL-12 albumin-binding domain fusion protein is passed in a continuous manner from the bioreactor to the purification step, thereby minimizing contact time with proteolytic enzymes secreted by cytokines that can cause clipping and degradation. In contrast, previous cytokine production methods often include a retention step in which the protein is held for a certain period before purification, which increases the exposure time to such proteolytic enzymes and also leads to proteolysis. Thus, the subject method advantageously enables high-yield production of cytokine-based products that exhibit less degradation than those produced using previous methods.

[0045] In some embodiments, not only viable host cells but also cell debris are prevented from leaving the bioreactor along with the culture medium containing the protein product. Any suitable technique can be used to prevent host cells from leaving the bioreactor. In certain embodiments, a filtration system is used to retain host cells from being removed along with the culture medium. Any suitable filtration system can be used that includes a membrane that retains cells and allows IL-12 ABD products to pass through the membrane. In exemplary embodiments, the filtration system is an ATF or TFF filtration system. In some embodiments, cells are retained in the bioreactor using gravity sedimentation, pumping through an internal filter, external loop flow through a filter, and centrifugation techniques.

[0046] In several embodiments, the culture medium containing the IL-12 ABD product is subjected to a purification system for purification for at least approximately 12 hours, at least approximately 18 hours, at least approximately 24 hours, at least approximately 30 hours, at least approximately 36 hours, at least approximately 42 hours, at least approximately 48 hours, at least approximately 54 hours, at least approximately 60 hours, at least approximately 66 hours, at least approximately 72 hours, at least approximately 78 hours, at least approximately 84 hours, at least approximately 90 hours, at least approximately 96 hours, at least approximately 102 hours, at least approximately 108 hours, at least approximately 114 hours, at least approximately 120 hours, at least approximately 126 hours, at least approximately 132 hours, and at least approximately 144 hours after the start of culture.

[0047] In some embodiments, the purification process takes place for at least about 6 hours, at least about 12 hours, at least about 18 hours, at least about 24 hours, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 3 weeks, or one month. In some embodiments, the culture medium containing the IL-12 ABD product is subjected to a purification system for purification if the cell viability of the culture medium is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%.

[0048] Protein products can be purified from spent culture media using any suitable chromatography column or combination thereof. Chromatographic columns that can be used in the method described in the subject include, but are not limited to, ion exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography, and affinity chromatography. In a preferred embodiment, the first column is directly and operably linked to a bioreactor, thereby enabling continuous downstream purification of protein products from spent culture media. In a particular embodiment, additional columns are connected to the first column in a stepwise manner.

[0049] In certain embodiments, a culture medium containing a protein product is subjected to at least one affinity chromatography column to further purify the protein product from contaminants containing proteolytic impurities. In affinity chromatography, target molecules are separated from the complex solution based on the affinity of the target molecule to a ligand or ligand-binding entity covalently bound to the matrix. Molecules in the complex solution or mixture that have little to no affinity for the ligand or ligand-binding entity flow unimpeded through the chromatography column, leaving the target molecule bound to the matrix. The target molecule can then be eluted from the chromatography column by modifying the buffer conditions to reduce the affinity of the target molecule to the ligand or ligand-binding entity. In certain embodiments, the chromatography material has the ability to selectively or specifically bind to the protein of interest. Non-limiting examples of such chromatography materials include: chromatography materials containing protein A, protein G, protein L, and molecules that bind to the protein product. In some embodiments, the method of the subject utilizes affinity chromatography containing a ligand or ligand-binding entity that has the ability to bind to the albumin-binding domain of an IL-12 ABD fusion protein. In some embodiments, affinity chromatography includes an antibody capable of binding to the albumin-binding domain of the IL-12 ABD fusion protein. In some embodiments, the antibody in the chromatography column has the ability to bind to the A10m3 albumin-binding domain (see Figure 4).

[0050] In some embodiments, the purification system includes a pseudoaffinity chromatography column. The pseudoaffinity column utilizes a dye as a ligand to bind to the target protein. In some embodiments, the pseudoaffinity column contains a ligand capable of binding to the target IL-12 ABD fusion protein (see Figure 5). The IL-12 ABD fusion protein is then subsequently captured by a ligand / serum albumin complex. In some embodiments, the pseudoaffinity column contains a cibacron blue ligand. In certain embodiments, the cibacron blue ligand is bound to an agarose-based matrix by a hydrophilic spacer. In some embodiments, the pseudoaffinity column is Capto® Blue resin (see Figure 14). The Capto® Blue resin is a cibacron blue ligand bound to an agarose-based matrix by a hydrophilic spacer.

[0051] In a particular embodiment, the culture medium containing the protein product is subjected to at least one ion exchange separation step, thereby obtaining an eluate containing the protein product. The ion exchange separation includes any method by which two substances are separated based on the difference in their respective ionic charges, and can employ either a cationic exchange material or an anionic exchange material.

[0052] The use of cationic or anionic exchange materials is based on the overall charge of the protein. Therefore, employing an anionic exchange step before using a cationic exchange step, or employing a cationic exchange step before using an anionic exchange step, is within the scope of the present invention. Furthermore, employing only a cationic exchange step, only anionic exchange steps, or any combination of the two in series is within the scope of the subject method.

[0053] In a particular embodiment, a culture medium containing a protein product is subjected to at least one size exclusion chromatography column. In size exclusion chromatography, the target molecule is separated from the complex solution or mixture based on size-related exclusion of the target molecule from the internal region of the spherical beads that make up the matrix. The progress of smaller molecules that have the ability to diffuse into the beads through the chromatography column is slowed with respect to the target molecule.

[0054] In certain embodiments, a culture medium containing a protein product is subjected to a hydrophobic interaction (HIC) chromatography column. In hydrophobic interaction chromatography, target molecules are separated from a complex solution or mixture based on the hydrophobicity of the target molecules. The complex solution containing the target molecules is applied to a chromatography column equilibrated with a high-salt buffer to promote the binding of the target molecules to the resin. A salt gradient mobile phase with reduced ionic strength is then introduced into the chromatography column to release the bound target molecules from the matrix. Alternatively, hydrophobic interaction chromatography can separate monomeric target molecules from a complex solution or mixture by binding hydrophobic impurities, including inactive dimers and aggregates of the target molecules, while allowing monomeric target molecules to flow relatively unimpeded through the chromatography column.

[0055] After purifying and recovering the protein product, the protein product is tested for purity. The purity of the protein product can be tested using any suitable technique. In some embodiments, the purity of the protein product is determined using SEC-HPLC, CE-SDS reduction, CE-IEF, glycan analysis by LC / MS, or RP-HPLC. In exemplary embodiments, the protein product has a purity of 65%, 70%, 75%, 80%, 85%, 90%, 95%, or greater than 99%.

[0056] All cited references are explicitly incorporated herein by reference in their entirety.

[0057] While specific embodiments of the present invention have been described above for illustrative purposes, it will be understood by those skilled in the art that numerous modifications of the details may be made without departing from the present invention as described in the appended claims. [Examples]

[0058] Examples illustrating the present invention are provided below. These examples are not intended to limit the present invention to any particular use or theory of operation.

[0059] IL-12 albumin-binding domain (ABD) fusion protein cell culture process The production of IL-12 albumin-binding domain fusion protein (IL-12 ABD) was carried out using a continuous process. IL-12 ABD contains an anti-albumin scFv called "A10m3" bound to single-stranded IL-12 (see Figure 5). For the continuous process, a perfusion-based system was used to establish highly efficient filtering using alternating tangential flow (ATF) through a hollow fiber filter. This mechanism allowed for cell growth with viability exceeding 85% at densities of up to 20 to 100 million cells per milliliter over a 25-day period. Upstream batches were carried out at various scales ranging from 2.5 L to 20 L in the manufacturing unit (GMP). In the 2.5 L batches, perfusion runs were carried out in a discontinuous manner but immediately processed to simulate a continuous process, while at the manufacturing scale, an inline capture step was performed downstream following the upstream perfusion. A bioreactor (manufacturer: Sartorius, Germany) equipped with the cell retention device ATF4 was loaded with the amount of cell culture medium (XtraCHO) required for a 2.5 L batch production, and ATF4 and ATF6 (manufacturer: Repligen, USA) were used during production (GMP). Dissolved oxygen concentration was controlled at 30 percent of air saturation (range 20–50 percent) by automatic correction of the gas composition entering through the headspace and intermittent sparging through ring spargers across all scales. The pH setpoint was 7.0 (range 6.8–7.4) and was also controlled by the flow of CO2 through spargers. Cells were placed at 1.2–1.8 × 10⁶ cells. 6 Inoculation live cell density range of cells / mL (Figure 1), and 1.5 × 10⁻⁶ 6Cells were inoculated into a bioreactor at a target seeding density of cells / mL. Using supply medium A (FMA) and supply medium B (FMB), the perfusion rate (Figure 1) was started at 0 hours with XtraCHO at 0.5 RV and increased as culture time and VCD increased (Figure 1). Samples collected from 0 to 96 hours were discarded, and the capture inline phase was started at 96 hours and continued until 120 hours (day 5), 144 hours (day 6), 168 hours (day 7), 192 hours (day 8), 216 hours (day 9), 240 hours (day 10), and 360 hours (day 15), as long as the viability was maintained above 85%. Small aliquots of the culture were collected every 24 hours, and parameters such as VCD (Figure 6A), viability (Figure 6A), residual glucose (Figure 6B), residual glutamine (Figure 7B), lactate (Figure 7A), ammonia levels (Figure 8A), and titer (Figure 8B) were checked. The bioreactor allowed for the collection of samples containing secreted proteins into the culture medium using an alternating tangential flow (ATF) filter, and a first chromatography system was connected to the bioreactor's surge bag for the purification of the collected recombinant therapeutic proteins. A summary of process parameters, as well as volume and cumulative productivity, is shown in Tables 1 and 2.

[0060] [Table 1]

[0061] [Table 2]

[0062] IL-12 albumin-binding domain (ABD) fusion protein purification process IL12-ABD molecules were captured from the bioreactor's ATF output by loading them onto an affinity-based chromatography column (Capto® Blue) connected to a Novasep automated continuous chromatography system. Capto® Blue resin was packed into the column, and the sample was loaded directly onto the column without any external modifications or treatments such as pH or dilution (Figure 9). Loading factors were obtained in the range of 0.7–2.5 mg / mL resin. The process could be operated with residence times ranging from 2.5–4 minutes, and was performed with a 4-minute residence time when batches were taken at production scale. Triton X-100 washing in the column was incorporated as a potential step for virus inactivation during the chromatography process (Figure 10). Process execution parameters are as follows (Table 3).

[0063] [Table 3]

[0064] The operating parameters for the chromatography process (flow rate, number of CVs passed, residence time, etc.) remained unchanged throughout the run, except for the number of cycles during the perfusion upstream process, when the feed concentration was increased in 0.03 mg / L increments up to 0.46 mg / mL of sample. The eluted fraction samples were analyzed offline for content and purity using RP-HPLC (representative profiles are shown in Figure 12). As can be seen from Figure 12, a significant amount of purification was observed after the initial capture step compared to the reference standard. The yield was calculated based on RP-UPLC data, considering the total protein in the elute fraction relative to the loaded protein, and an average recovery of 66% was achieved (Figure 11). For further control on the impurity profile, the elute from chromatography 1 was treated on phenyl Sepharose fast-flow high-sub resin (hydrophobic interaction chromatography) followed by POROS XS resin (cation exchange chromatography). The final drug substance was prepared using UFDF, followed by cation exchange chromatography and nanofiltration, and then sterile filtration. Next, the final active pharmaceutical ingredient was compared to a reference standard using RP-HPLC and SE-HPLC methods. The operational process flow and parameters used for the production scale batch performed on the IL-12 ABD fusion protein are shown in Figure 9.

[0065] The Capto® Blue affinity column was also tested for the purification of the IL-12 / IL-15 ABD fusion protein (1210) containing the A10m3 albumin-binding domain. However, unlike the IL-12 ABD fusion protein, Capto® Blue did not selectively bind to the IL-12 / IL-15 ABD fusion protein under any of the tested elution conditions and was present in all elution fractions (see Figures 12 vs. 13).

[0066] The embodiments described above are provided to give a complete disclosure and description of methods for preparing and using embodiments of the compositions, systems, and methods of the present invention to those skilled in the art, and are not intended to limit the scope of what the inventors consider to be their own invention. Modifications of the modes described above for carrying out the invention, which would be obvious to those skilled in the art, are intended to be within the scope of the following claims. All patents and publications mentioned herein indicate the level of skill of those skilled in the art in the field to which the invention relates.

[0067] All headings and section designations are used solely for clarity and reference purposes and are not intended to limit them in any sense. For example, those skilled in the art will understand the usefulness of combining various aspects from different headings and sections as needed, in accordance with the spirit and scope of the invention described herein.

[0068] All references cited herein are incorporated herein by reference in whole and for all purposes, just as each individual publication or patent or patent application is specifically and individually indicated to be incorporated by reference in whole for all purposes.

[0069] As will be apparent to those skilled in the art, many modifications and variations of this application can be made without departing from its spirit and scope. The specific embodiments and examples described herein are provided for illustrative purposes only, and this application, along with the entire scope of equivalents to which the claims are entitled, is limited only by the conditions of the appended claims.

Claims

1. A method for producing proteins, a) A step of culturing a plurality of mammalian host cells, each containing a polynucleotide encoding the protein, in a bioreactor containing a liquid culture medium, The steps include culturing the host cells under conditions in which the protein is produced and secreted into the liquid culture medium by the host cells, b) A step of removing the liquid culture medium containing the secreted protein from the bioreactor, c) A step of purifying the protein by passing the liquid culture medium removed from the bioreactor directly through one or more chromatography columns, A purification step in which one or more chromatography columns are operably and continuously connected to a perfusion bioreactor, d) A step of recovering the purified protein from the liquid culture medium, Includes, A method wherein the protein is an IL-12 albumin-binding domain (ABD) fusion protein.

2. The method according to claim 1, wherein the bioreactor is a perfusion-mode bioreactor.

3. The method according to claim 2, wherein the liquid culture medium containing the secreted protein is removed from the perfusion-mode bioreactor and replaced with fresh liquid culture medium at set time intervals.

4. The method according to claim 3, wherein the set time interval is 12 hours, 24 hours, 48 ​​hours, or 72 hours.

5. The method according to claim 2, wherein the liquid culture medium containing the secreted protein is continuously removed from the perfusion-mode bioreactor and continuously replaced with fresh liquid culture medium.

6. The method according to any one of claims 3 to 5, wherein the liquid culture medium is removed from the bioreactor at least about 96 hours after the start of the culture step a).

7. The method according to any one of claims 3 to 6, wherein the liquid culture medium containing the secreted protein is removed from the perfusion-mode bioreactor and replaced with an equal volume of fresh liquid culture medium.

8. The method according to claim 3 or 4, wherein a portion of the volume of the liquid culture medium containing the secreted protein is partially removed from the perfusion-mode bioreactor and replaced with an equal volume of fresh liquid culture medium.

9. The method according to any one of claims 1 to 8, wherein the host cells are prevented from leaving the perfusion bioreactor during removal step b) using a cell separation system.

10. The method according to any one of claims 1 to 9, wherein the host cells are cultured in the presence of nutrients that are periodically replenished in the liquid culture medium.

11. The aforementioned multiple mammalian host cells are subjected to a 20-100 × 10⁶ temperature in the bioreactor. ６ The method according to any one of claims 1 to 10, wherein the cells are cultured in a liquid culture medium at a density of cells / mL.

12. The method according to any one of claims 1 to 11, wherein the purification step c) separates one or more proteolytic enzymes from the protein.

13. The method according to any one of claims 1 to 12, wherein the albumin-binding domain of the IL-12 ABD fusion protein comprises a variable heavy chain domain or a variant thereof containing SEQ ID NO: 1, and a variable light chain domain or a variant thereof containing SEQ ID NO:

5.

14. The method according to claim 13, wherein the albumin-binding domain is an scFv comprising the amino acid sequence of SEQ ID NO: 9 or a variant thereof.

15. The method according to any one of claims 1 to 14, wherein the IL-12 of the IL-12 fusion protein is a single-stranded IL-12 containing a p35 subunit covalently bonded to a p40 subunit.

16. The method according to claim 15, wherein the p40 subunit has the amino acid sequence of SEQ ID NO: 10 or a variant thereof.

17. The method according to claim 15 or 16, wherein the p35 subunit has the amino acid sequence of SEQ ID NO: 11 or a variant thereof.

18. The method according to any one of claims 15 to 17, wherein the single-stranded IL-12 has the amino acid sequence of SEQ ID NO:

12.

19. The method according to any one of claims 1 to 18, wherein the IL-12 ABD fusion protein has the amino acid sequence of SEQ ID NO:

13.

20. The method according to any one of claims 1 to 19, wherein the one or more chromatography columns include an ion exchange chromatography column, a hydrophobic interaction column, an affinity column, a pseudoaffinity column, or a size exclusion chromatography column.

21. The method according to claim 20, wherein one or more chromatography columns include affinity columns.

22. The method according to claim 21, wherein the pseudo-affinity column contains a cibacron blue ligand.

23. The method according to claim 22, wherein the pseudo-affinity column includes the affinity chromatography medium shown in Figure 14.

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