Methods for Producing Fusion Polypeptides

Abstract

The present disclosure provides surprisingly useful methods for making fusion polypeptides comprising an immunomodulatory moiety and a metal hydroxide-binding moiety, as well as a variety of related technologies, including methods for making fusion polypeptide-metal hydroxide complexes and methods for using such fusion polypeptides and / or fusion polypeptide-metal hydroxide complexes.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and benefit of U.S. Provisional Patent Application No. 63 / 424,047, filed November 9, 2022, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] Useful fusion polypeptides have been developed in which metal-binding polypeptides are conjugated to immunomodulatory domains (e.g., IL-12 immunomodulatory domains) (see, e.g., published international patent application WO2020 / 263399). Particular uses of such fusion peptides include the treatment of certain medical conditions, such as cancer. Summary of the Invention

[0003] The present disclosure provides certain methods for producing fusion polypeptides and preparations that specifically include such, for example, IL-12-fusion polypeptides (e.g., as described in published international patent application WO 2020 / 263399). In some embodiments, the provided techniques achieve efficient and effective generation of preparations of phosphorylated forms of such fusion polypeptides.

[0004] Among other things, the present disclosure identifies a source of problems with certain manufacturing techniques utilized to prepare related fusion polypeptides (e.g., fusion polypeptides in which a metal-binding polypeptide is conjugated to an immunomodulatory domain, e.g., an IL-12 immunomodulatory domain, and particularly phosphorylated form(s) of such fusion polypeptide(s). Without being bound by any particular theory, the present disclosure notes that phosphorylated forms of fusion polypeptides can contribute to and / or cause certain such manufacturing challenges. The present disclosure provides solutions to this identified problem, including particularly useful methods for producing phosphorylated forms of fusion polypeptides. In some embodiments, such provided methods may include co-expressing a fusion polypeptide with a kinase in a host cell such that the fusion polypeptide and kinase are expressed at a ratio ranging from about 4:1 to about 10:1. In some embodiments, the provided methods achieve the production of phosphorylated fusion polypeptide preparations characterized by a degree of phosphorylation (e.g., average number of phosphate molecules per polypeptide) of about 7-9. Without being bound by any particular theory, the present disclosure notes that excessively low phosphorylation may result in weaker binding to the metal hydroxide backbone and potentially increased systemic leakage after administration of the cytokine:metal hydroxide complex to a patient; conversely, excessively high phosphorylation may result in reduced cytokine potency after binding to the metal hydroxide backbone, potentially due to structural changes. In some embodiments, methods according to the present disclosure provide (e.g., achieve production of preparation(s) thereof) phosphorylated forms of fusion polypeptides with improved metal hydroxide (e.g., alum) conjugation.

[0005] Among other things, the present disclosure provides the surprising discovery that improved methods for producing phosphorylated forms of fusion polypeptides can be developed (e.g., compared to methods that use affinity chromatography capture steps and / or that use host cells in which the expression ratio of fusion polypeptide and kinase is not within the range of about 4:1 to about 10:1).

[0006] In particular, the present disclosure demonstrates that typical methods of producing phosphorylated form(s) of fusion polypeptides via affinity chromatography do not achieve the degree(s) of phosphorylation described herein. Despite art-recognized expectations that affinity chromatography may be desirable or even necessary to effectively isolate phosphorylated form(s) of the relevant fusion polypeptide(s), the present disclosure surprisingly demonstrates that the provided methods, lacking any affinity chromatography step, can achieve superior results.

[0007] The present disclosure recognizes that methods including affinity chromatography steps can have various drawbacks, such as, for example, lengthy and / or expensive purification processes that typically involve multiple steps, which together can result in significant loss of product recovery. Moreover, the present disclosure demonstrates that the provided methodologies can achieve improved product quality (e.g., reduced levels of one or more unwanted species, e.g., product-associated species), and / or reduced impurity load (e.g., host cell proteins, DNA) when compared to certain conventional methods, including methods that include one or more affinity chromatography steps.

[0008] The present disclosure provides particularly useful methods for achieving the production of highly pure preparations of the phosphorylated fusion polypeptides described herein. The provided techniques offer improvements over and / or solve problems associated with various conventional production techniques. In certain embodiments, the provided production methods (e.g., particularly provided purification methods) do not include an affinity chromatography capture step; alternatively or additionally, in some embodiments, the provided production methods (e.g., particularly provided purification methods) include one or more purification steps selected from an anion chromatography capture step; and a hydrophobic interaction chromatography refinement step. In some embodiments, the provided techniques involve an anion chromatography step followed (e.g., immediately following) a hydrophobic interaction step. In some embodiments, the hydrophobic interaction step is followed by a second anion chromatography step. In certain embodiments, such provided techniques are applied to, for example, cell extracts that can be prepared from engineered mammalian cells provided by the present disclosure (e.g., engineered mammalian cells expressing a fusion polypeptide described herein and a kinase that phosphorylates such fusion polypeptide, e.g., engineered mammalian cells expressing the fusion polypeptide and kinase in a ratio ranging from about 4:1 to about 10:1, e.g., about 8:1). [Brief explanation of the drawings]

[0009] [Figure 1] 1 shows an exemplary schematic diagram of a fusion polypeptide-metal hydroxide complex of the present disclosure, which can be administered to a subject, resulting in increased retention and / or efficacy compared to an appropriate reference standard. [Figure 2] 1 shows a diagram of an exemplary fusion polypeptide of the disclosure comprising a first (p40) and a second (p35) IL12 immunoagonist moiety and a metal hydroxide binding polypeptide with multiple phosphorylation sites. ABP: Alum-binding peptide. [Figure 3A] Viability during a 1 L fed-batch run of stable pools is shown. [Figure 3B]Viable cell density (VCD) during a 1 L fed-batch run of stable pools is shown. [Figure 3C] 1 shows the qualitative analysis of IL12 fusion proteins by non-reducing sodium dodecyl polyacrylamide gel electrophoresis (NR SDS-PAGE). [Figure 4A] Figure 1 shows the viability during a 1 L fed-batch run of stable pools representing different ratios of IL12 fusion protein to FAM20C. [Figure 4B] Figure 1 shows the viable cell density in a 1 L fed-batch run of stable pools representing different ratios of IL12 fusion protein to FAM20C: P1: Pool 6880 D208_8:1; P2: Pool 6881 D208_15:1; P3: Pool 6882 D211_8:1; P4: Pool 6883 D211_15:1; P5: Pool 6884 D206_8:1; and P6: Pool 6885 D209_8:1. [Figure 5] Figure 1 shows the productivity (titer) during 1 L fed-batch runs of stable pools representing different ratios of IL12 fusion protein to FAM20C as assessed by biolayer interferometry (BLI). [Figure 6] The viability of stable pool 6880 P1 (8:1) after transfection and corresponding recovery is shown. [Figure 7A] Figure 1 shows the viability of stable pool 6880 P1 (8:1) in a 1 L fed-batch culture. [Figure 7B] Figure 1 shows the viable cell density in a 1 L fed-batch culture of stable pool 6880 P1 (8:1). [Figure 7C] The titer of stable pool 6880 P1 (8:1) in a 1 L fed-batch culture is shown. [Figure 8] Figure 1 shows the full chromatogram and zoom of the elution peak during the first chromatography of the clarified harvest of stable pool 6880 P1 by anion exchange chromatography (GigaCap Q). [Figure 9]Qualitative analysis of anion exchange chromatography elution fractions by non-reducing SDS-PAGE is shown. Fractions 13-25 were pooled. The volume was 18 mL, and the concentration was 6.57 mg / mL. [Figure 10] 1 shows the full chromatogram and zoom of the elution peak during the first chromatography of the clarified harvest of stable pool 6880 P1 by hydroxyapatite chromatography as the second chromatography step. [Figure 11] Qualitative analysis of hydroxyapatite chromatography fractions of 6880 P1 on non-reducing SDS-PAGE. 3µg per lane. FT1 (lane 14) was acquired in-line mid-load. FT2 (lane 15) was acquired in-line at the end of load (maximum absorbance). Pool A: Fractions 6-9 from each run. 10mL at 2.63mg / mL. Pool B: Fractions 10-13 from each run. 10mL at 1.30mg / mL. [Figure 12] 1 shows the qualitative analysis of the hydrophobic interaction chromatography (phenyl) fraction of 6880 P1 on non-reducing SDS-PAGE as a second chromatographic step. [Figure 13A] Chromatographic (A) and qualitative analysis of phenyl chromatography fractions of 6880 P1 on non-reducing SDS-PAGE. [Figure 13B] The purity of the eluted fractions is determined by analytical size exclusion chromatography (SEC-HPLC) (B). [Figure 13C] Proteins eluted with 0.5 M ammonium sulfate are shown by dotted lines, or proteins eluted with 0.75 M followed by 0.5 M ammonium sulfate are shown by solid lines. [Figure 14]Qualitative analysis of heparin affinity and hydrophobic interaction chromatography (phenyl) fractions of 6880 P1 (when both were used as the second chromatography step) on non-reducing SDS-PAGE. Non-reducing, stain-free 4-20%, 3µg per lane. Lane 2: Heparin 0.1M E1, Lane 3: Heparin 0.1M E2, Lane 4: Heparin 0.35M E1, Lane 5: Heparin 0.35M E2, Lane 6: Heparin 2M E1, Lane 7: Phenyl 0.75M E1, Lane 8: Phenyl 0M E2, Lane 9: QHP load. [Figure 15] Qualitative analysis of Q HP anion exchange chromatography fractions of 6880 P1 on non-reducing SDS-PAGE when used as a third chromatography step. Non-reducing, stain-free 4-20%, 3µg per lane. Lane 2: Strip, Lane 3: Fraction 397, Lane 4: Fraction 404, Lane 5: Fraction 409, Lane 6: Fraction 414, Lane 7: Fraction 419, Lane 8: Fraction 424, Lane 9: Phenylload. [Figure 16A] Shown are purity analyses of heparin chromatography fractions (upper panel in A), phenyl chromatography fractions (lower panel in A), and Q HP anion exchange chromatography fractions (B) of 6880 P1 by SEC-HPLC when used as the first, second, and third chromatography steps, respectively. [Figure 16B] Shown are purity analyses of heparin chromatography fractions (upper panel in A), phenyl chromatography fractions (lower panel in A), and Q HP anion exchange chromatography fractions (B) of 6880 P1 by SEC-HPLC when used as the first, second, and third chromatography steps, respectively. [Figure 17]1 shows the chromatogram of ANK101 (IL-12 fusion polypeptide) refined after TOYOPEARL GigaCap Q-650S chromatogram during multiple cycles. The blue line is the A280 nm trace for cycle 9, the orange line is the A280 nm trace for cycle 10, the green line is the A280 nm trace for cycle 11, and the purple line is the A280 nm trace for cycle 12. [Figure 18] 1 shows overview chromatograms of reduced RP HPLC profiles for an IL-12 fusion polypeptide reference standard (top panel) and an IL-12 fusion polypeptide GMP drug substance batch (bottom panel). [Figure 19] 1 shows overview chromatograms of reduced CE-SDS electropherograms for an IL-12 fusion polypeptide reference standard (top panel) and a GMP drug substance batch (bottom panel). [Figure 20] Shown are full SE HPLC chromatograms for the IL-12 fusion polypeptide reference standard (top panel) and the GMP drug substance batch (bottom panel). [Figure 21] Shown are overviews of AEX HPLC chromatograms for the IL-12 fusion polypeptide reference standard (top panel) and the GMP drug substance batch (bottom panel). [Figure 22] Zoomed-in views of deglycosylated ESI-MS processed spectral profiles of IL-12 fusion polypeptide drug substance in-process samples before excipient (polysorbate 20) addition are shown for the reference standard (top panel) and GMP drug substance batch (bottom panel). [Figure 23] Total ion current chromatograms are shown for the drug product GMP batch (top panel), the reference standard (middle panel), and the deglycosylated sample for the assay control (NEC) (bottom panel). The peak at RT=16.5 min corresponds to a mass of approximately 34,778 Da and was assigned to PNGaseF. Only the major peak was considered for data collection. [Figure 24-1]Deconvoluted MS spectra of the reference standard (lower graph) and reduced and deglycosylated sample for the drug product GMP batch (upper graph) are shown. [Figure 24-2] Same as above [Figure 25-1] An overview of the neutral N-linked oligosaccharide profile of the IL-12 fusion polypeptide reference standard (upper panel / P4130826ARS) and the GMP drug substance batch (lower panel / 1205114) is shown. [Figure 25-2] Same as above DETAILED DESCRIPTION OF THE INVENTION

[0010] definition Administration: As used herein, the term "administration" typically refers to the application of a composition to a subject or system. Those of skill in the art will recognize various routes that may be utilized for administration to a subject, e.g., a human, in appropriate circumstances. For example, in some embodiments, administration may be systemic; in some embodiments, administration may be local. In some embodiments, administration may be enteral; in some embodiments, administration may be parenteral. In some embodiments, administration may be by injection (e.g., intramuscular, intratumoral, intravenous, or subcutaneous injection). In some embodiments, injection may include bolus injection, infusion, perfusion, or infusion. In many embodiments, administration according to the present disclosure is by intratumoral injection.

[0011] Affinity: As known in the art, "affinity" is a measure of the tightness with which two or more binding partners bind to one another. Those skilled in the art will be knowledgeable about various assays that can be used to assess affinity and will also recognize appropriate controls for such assays. In some embodiments, affinity is assessed in a quantitative assay. In some embodiments, affinity (e.g., of one binding partner at a time) is assessed across multiple concentrations. In some embodiments, affinity is assessed in the presence of one or more potential competitors (e.g., that may be present in a relevant physiological situation). In some embodiments, affinity is assessed relative to a reference (e.g., having a known affinity above a certain threshold [see "positive control"] or having a known affinity below a certain threshold [see "negative control"]). In some embodiments, affinity may be assessed relative to a concurrent reference, and in some embodiments, affinity may be assessed relative to a background reference. Typically, when affinity is assessed relative to a reference, it is assessed under comparable conditions.

[0012] Agent: Generally, as used herein, the term "agent" is used to refer to an entity (e.g., a lipid, metal, nucleic acid, polypeptide, polysaccharide, small molecule, etc., or a complex, combination, mixture, or system thereof (e.g., a cell, tissue, organism)) or a phenomenon (e.g., heat, an electric current or electric field, a magnetic force or field, etc.). Under appropriate circumstances, as will be clear from the context to one of skill in the art, the term may be used to refer to an entity that is or includes a cell or organism, or a fraction, extract, or component thereof. Alternatively, or additionally, as will be clear from the context, the term may be used to refer to a natural product, in the sense of being found in nature and / or obtained from nature. In some cases, also as will be clear from the context, the term may be used to refer to one or more entities that are man-made, in the sense of being designed, engineered, and / or produced by the hand of man and / or not found in nature. In some embodiments, an agent may be utilized in isolated or pure form, and in some embodiments, an agent may be utilized in crude form. In some embodiments, potential agents may be provided as collections or libraries, eg, that can be screened to identify or analyze active agents contained therein.

[0013] Agonist: Those skilled in the art will understand that the term "agonist" can be used to refer to an agent, condition, or event whose presence, level, degree, type, or form correlates with an increase in the level or activity of another agent (i.e., a stimulated agent or a target agent). Generally, an agonist can be or include an agent of any chemical class, such as, for example, a small molecule, a polypeptide, a nucleic acid, a carbohydrate, a lipid, a metal, and / or any other entity that exhibits related activation activity. In some embodiments, an agonist can be direct (e.g., when it exerts its effect directly on a target by physically binding to such target); in some embodiments, an agonist can be indirect (when it exerts its effect by other than binding to its target; e.g., by interacting with a regulator of the target such that the level and / or activity of the target is altered).

[0014] Amino acid: In its broadest sense, the term "amino acid" as used herein refers to compounds and / or substances that can be, are, or are incorporated into a polypeptide chain, for example, by the formation of one or more peptide bonds. In some embodiments, an amino acid has the general structure HN-C(H)(R)-COOH. In some embodiments, an amino acid is a naturally occurring amino acid. In some embodiments, an amino acid is a non-naturally occurring amino acid. In some embodiments, an amino acid is a D-amino acid. In some embodiments, an amino acid is an L-amino acid. A "standard amino acid" refers to any of the 20 standard L-amino acids commonly found in naturally occurring peptides. A "non-standard amino acid" refers to any amino acid other than the standard amino acids, whether synthetically prepared or obtained from a natural source. In some embodiments, amino acids, including the carboxy-terminal amino acid and / or the amino-terminal amino acid in a polypeptide, may contain structural modifications compared to the above general structure. For example, in some embodiments, an amino acid may be modified relative to the general structure by methylation, amidation, acetylation, pegylation, glycosylation, phosphorylation, and / or substitution (e.g., of an amino group, a carboxylic acid group, one or more protons, and / or a hydroxyl group). In some embodiments, such modifications may, for example, alter the circulating half-life of a polypeptide comprising the modified amino acid compared to one comprising the otherwise identical amino acid. In some embodiments, such modifications do not significantly alter the relevant activity of a polypeptide comprising the modified amino acid compared to one comprising the otherwise identical amino acid. As will be clear from the context, in some embodiments, the term "amino acid" may be used to refer to a free amino acid; in some embodiments, the term may be used to refer to an amino acid residue of a polypeptide.

[0015] Animal: As used herein, the term "animal" refers to a member of the animal kingdom. In some embodiments, "animal" refers to a human of either sex and at any stage of development. In some embodiments, "animal" refers to a non-human animal at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, mouse, rat, rabbit, monkey, dog, cat, horse, sheep, cow, primate, and / or pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and / or parasites. In some embodiments, the animal may be a transgenic animal, a genetically modified animal, and / or a clone.

[0016] Binding: As used herein, the term "binding" is generally understood to mean a non-covalent association between two or more entities. "Direct" binding involves physical contact between the entities or moieties, while indirect binding involves a physical interaction due to physical contact through one or more intermediate entities. Binding between two or more entities can typically be assessed in any of a variety of contexts, including when the interacting entities or moieties are studied alone or in the context of a more complex system (e.g., covalently, electrostatically, or otherwise associated with a carrier entity, and / or in a biological system or cell). Binding between two entities can be considered "specific" if, under the conditions being assessed, the associated entities are more likely to associate with each other than with other available binding partners.

[0017] Buffer: As used herein, the term "buffer" typically refers to a solution that will cause a change in pH based on the amount of acid or alkali in it. A buffer resists changes in pH due to the presence of its acid-base components. Typical buffers utilized in biological manufacturing processes maintain pH within the physiological range. Traditional buffer components include, but are not limited to, organic and inorganic salts, acids and bases. Commonly used buffers include, but are not limited to, tricine and bicine, MES, PIPES, HEPES, MOPS, and PBS.

[0018] Cancer: The terms "cancer," "malignancy," "neoplasm," "tumor," and "carcinoma" are used herein to refer to cells that exhibit relatively abnormal, uncontrolled, and / or autonomous growth, resulting in an aberrant growth phenotype characterized by a significant loss of control of cell proliferation. In some embodiments, tumors may be or include pre-cancerous (e.g., benign), malignant, pre-metastatic, metastatic, and / or non-metastatic cells. The present disclosure specifically identifies particular cancers to which its teachings may be particularly relevant. In some embodiments, the relevant cancers may be characterized as solid tumors. In some embodiments, the relevant cancers may be characterized as hematological tumors. In general, examples of various types of cancer known in the art include, for example, hematopoietic cancers including leukemia, lymphoma (Hodgkin's lymphoma and non-Hodgkin's lymphoma), myeloma and myeloproliferative disorders; sarcomas, melanomas, adenomas, and carcinomas of solid tissue; squamous cell carcinoma of the mouth, throat, larynx, and lung; liver cancer; genitourinary cancers such as prostate cancer, cervical cancer, bladder cancer, uterine cancer, and endometrial cancer, and renal cell carcinoma; bone cancer; pancreatic cancer; skin cancer; cutaneous or intraocular melanoma; cancers of the endocrine system; cancer of the thyroid gland; cancer of the parathyroid gland; head and neck cancer; breast cancer; gastrointestinal cancer; cancers of the nervous system; benign lesions such as papilloma, etc.

[0019] Characteristic sequence element: As used herein, the phrase "characteristic sequence element" refers to a sequence element found in a polymer (e.g., a polypeptide or nucleic acid) that represents a characteristic portion of that polymer. In some embodiments, the presence of a characteristic sequence element correlates with the presence or level of a particular activity or property of the polymer. In some embodiments, the presence (or absence) of a characteristic sequence element defines a particular polymer as a member (or not) of a particular family or group of such polymers. A characteristic sequence element typically comprises at least two monomers (e.g., amino acids or nucleotides). In some embodiments, a characteristic sequence element comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, or more monomers (e.g., sequentially linked monomers). In some embodiments, a characteristic sequence element comprises at least first and second stretches of consecutive monomers separated by one or more spacer regions that may or may not vary in length between polymers sharing the sequence element.

[0020] Chemotherapeutic Agent: The term "chemotherapeutic agent," as used herein, has its art-understood meaning and refers, inter alia, to one or more pro-apoptotic, cytostatic, and / or cytotoxic agents, including agents available and / or recommended for use in the treatment of one or more diseases, disorders, or conditions associated with unwanted cell proliferation. In many embodiments, chemotherapeutic agents are beneficial in the treatment of cancer. In some embodiments, the chemotherapeutic agent may be or include one or more alkylating agents, one or more anthracyclines, one or more cytoskeletal disruptors (e.g., microtubule-targeting agents such as taxanes, mei-taisin, and analogs thereof), one or more epothilones, one or more histone deacetylase (HDAC) inhibitors, one or more topoisomerase inhibitors (e.g., inhibitors of topoisomerase I and / or topoisomerase II), one or more kinase inhibitors, one or more nucleotide analogs or nucleotide precursor analogs, one or more peptide antibiotics, one or more platinum-based agents, one or more retinoids, one or more vinca alkaloids, and / or one or more analogs of one or more of the following (i.e., those sharing related antiproliferative activity): In some particular embodiments, the chemotherapeutic agent is actinomycin, all-trans retinoic acid, auristatin, azacitidine, azathioprine, bleomycin, bortezomib, carboplatin, capecitabine, cisplatin, chlorambucil, cyclophosphamide, curcumin, cytarabine, daunorubicin, docetaxel, doxifluridine, doxorubicin, epirubicin, epothilone, etoposide, fluorouracil, gemcitabine, hydroxyurea, The chemotherapeutic agent may be or include one or more of idarubicin, imatinib, irinotecan, meytaicin and / or its analogs (e.g., DM1), mechlorethamine, mercaptopurine, methotrexate, mitoxantrone, maytansinoids, oxaliplatin, paclitaxel, pemetrexed, teniposide, thioguanine, topotecan, valrubicin, vinblastine, vincristine, vindesine, vinorelbine, and combinations thereof. In some embodiments, the chemotherapeutic agent may be utilized in conjunction with an antibody-drug conjugate.In some embodiments, the chemotherapeutic agent is hLL1-doxorubicin, hRS7-SN-38, hMN-14-SN-38, hLL2-SN-38, hA20-SN-38, hPAM4-SN-38, hLL1-SN-38, hRS7-Pro-2-P-Dox, hMN-14-Pro-2-P-Dox, hLL2-Pro-2-P-Dox, hA20-Pro-2-P-Dox, hPAM4-Pro-2-P-Dox, hLL1-Pro-2-P-Dox, P 4 / D10-doxorubicin, gemtuzumab ozogamicin, brentuximab vedotin, trastuzumab emtansine, inotuzumab ozogamicin, glembatumomab vedotin, SAR3419, SAR566658, BIIB015, BT062, SGN-75, SGN-CD19A, AMG-172, AMG-595, BAY-94-9343, ASG-5ME, ASG-22ME, ASG-16M8F, MDX-1203, MLN-0264, anti-PSMA ADC, RG-7450, RG-7458, RG-7593, RG-7596, RG-7598, RG-7599, RG-7600, RG-7636, ABT-414, IMGN-853, IMGN-529, borsetuzumab mafodotin, and lorvotuzumab mertansine. In some embodiments, the chemotherapeutic agent can be one described as being utilized in an antibody-drug conjugate as described or discussed in one or more of the following: Govindan et al., The Scientific World Journal 10:2070, 2010,-2089. In some embodiments, the chemotherapeutic agent may be or include one or more of farnesyl-thiosalicylic acid (FTS), 4-(4-chloro-2-methylphenoxy)-N-hydroxybutanamide (CMH), estradiol (E2), tetramethoxystilbene (TMS), δ-tocatrienol, salinomycin, or curcumin.

[0021] Combination therapy: As used herein, the term "combination therapy" refers to those situations in which a subject is exposed to two or more therapeutic regimens (e.g., two or more therapeutic agents) simultaneously. In some embodiments, the two or more regimens may be administered simultaneously. In some embodiments, the regimens may be administered sequentially (e.g., all "doses" of a first regimen are administered before any doses of a second regimen are administered). In some embodiments, the therapeutic agents are administered in overlapping dosing regimens. In some embodiments, "administration" of a combination therapy may include administering one or more therapeutic agent(s) or modalities to a subject receiving other therapeutic agent(s) or modalities in conjunction. For clarity, combination therapy does not require that individual therapeutic agents be administered together in a single composition (or even necessarily simultaneously), although in some embodiments, two or more therapeutic agents, or active portions thereof, may be administered together in a combination composition or even in a combination compound (e.g., as part of a single chemical complex or covalent entity).

[0022] Dosing regimen: Those skilled in the art will understand that the term "dosing regimen" can refer to a set of unit doses (typically more than one) administered individually to a subject, typically spaced apart. In some embodiments, a given therapeutic agent has a recommended dosing regimen that can include one or more doses. In some embodiments, a dosing regimen includes multiple doses, each separated in time from the other doses. In some embodiments, the individual doses are separated from each other by periods of equal length, and in some embodiments, a dosing regimen includes multiple doses and at least two different periods separating the individual doses. In some embodiments, all doses within a dosing regimen are the same unit dose amount. In some embodiments, different doses within a dosing regimen are of different amounts. In some embodiments, a dosing regimen includes a first dose in a first dose amount, followed by one or more additional doses in a second dose amount that is different from the amount of the first dose. In some embodiments, the dosing regimen comprises a first dose in a first dosage amount, followed by one or more additional doses in a second dosage amount that is the same as the first dosage amount. In some embodiments, the dosing regimen is correlated with a desired or beneficial outcome when administered across a relevant population (i.e., is a therapeutic dosing regimen).

[0023] Epitope: As used herein, the term "epitope" refers to a moiety that is specifically recognized by an immunoglobulin (e.g., antibody or receptor) binding entity. In some embodiments, an epitope is composed of multiple chemical atoms or groups on an antigen. In some embodiments, such chemical atoms or groups are surface-exposed when the antigen adopts a related three-dimensional structure. In some embodiments, such chemical atoms or groups are physically close to each other in space when the antigen adopts such a conformation. In some embodiments, at least some such chemical atoms or groups are physically separated from each other when the antigen adopts an alternative conformation (e.g., linearized).

[0024] Excipient: As used herein, refers to a non-therapeutic agent that may be included in a pharmaceutical composition, for example, to provide or contribute to a desired consistency or stabilizing effect. Suitable pharmaceutical excipients include, for example, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, etc.

[0025] Expression: As used herein, the term "expression" of a nucleic acid sequence refers to the production of any gene product from the nucleic acid sequence. In some embodiments, the gene product can be a transcription product. In some embodiments, the gene product can be a polypeptide. In some embodiments, expression of a nucleic acid sequence involves one or more of: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of the RNA transcript (e.g., by splicing, editing, etc.); (3) translation of the RNA into a polypeptide or protein; and / or (4) post-translational modification of the polypeptide or protein.

[0026] Functional: As used herein, the term "functional" is used to refer to forms or fragments of an entity that exhibit particular properties and / or activities.

[0027] Fragment: A "fragment" of a material or entity described herein comprises a discrete portion of the whole, but has a structure that lacks one or more portions found in the whole. In some embodiments, the fragment consists of such a discrete portion. In some embodiments, the fragment consists of or comprises a characteristic structural element or portion found in the whole. In some embodiments, a fragment of a polymer comprises or consists of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more monomeric units (e.g., residues) present in the entire polymer. In some embodiments, a polymeric fragment comprises or consists of at least about 5%, 10%, 15%, 20%, 25%, 30%, 25%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of the monomeric units (e.g., residues) found in the whole polymer. The whole substance or entity may, in some embodiments, be referred to as the "parent" of the fragment.

[0028] Gene: As used herein, the term "gene" refers to a DNA sequence in a chromosome that encodes a product (e.g., an RNA product and / or a polypeptide product). In some embodiments, a gene includes coding sequence (i.e., a sequence that encodes a specific product), and in some embodiments, a gene includes non-coding sequence. In some particular embodiments, a gene can include both coding (e.g., exon) and non-coding (e.g., intron) sequence. In some embodiments, a gene can include one or more regulatory elements that can, for example, control or influence one or more aspects of gene expression (e.g., cell-type specific expression, inducible expression, etc.).

[0029] Gene product or expression product: As used herein, the term "gene product" or "expression product" generally refers to the RNA transcribed from a gene (before and / or after processing) or the polypeptide (before and / or after modification) encoded by the RNA transcribed from a gene.

[0030] Genome: As used herein, the term "genome" means the total genetic information contained in an individual organism or cell and represented by the complete DNA sequence of its chromosomes.

[0031] High molecular weight species (HMWS): As used herein, refers to a dimer or multimer of a polypeptide (e.g., a fusion polypeptide). Such species can be identified, for example, by size exclusion chromatography (SEC)-HPLC.

[0032] Host cell: As used herein, refers to a cell into which exogenous DNA (recombinant or otherwise) has been introduced. Those skilled in the art will understand, upon reading this disclosure, that such terms refer not only to the particular subject cell but also to the progeny of such a cell. Because certain modifications may occur in subsequent generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein. In some embodiments, host cells include prokaryotic and eukaryotic cells selected from any of the kingdoms of life suitable for expressing exogenous DNA (e.g., recombinant nucleic acid sequences). Exemplary cells include prokaryotic and eukaryotic (unicellular or multicellular), bacterial cells (e.g., strains of E. coli, Bacillus spp., Streptomyces spp., etc.), mycobacterial cells, fungal cells, yeast cells (e.g., S. cerevisiae, S. pombe, P. pastoris, P. methanolica, etc.), plant cells, insect cells (e.g., SF-9, SF-21, baculovirus-infected insect cells, Trichoplusia ni, etc.), non-human animal cells, human cells, or cell fusions, e.g., hybridomas or quadromas. In some embodiments, the cells are human, monkey, ape, hamster, rat, or mouse cells. In some embodiments, the cell is a eukaryotic cell and is selected from the following cells: CHO (e.g., CHO K1, DXB-1 1 CHO, Veggie-CHO), COS (e.g., COS-7), retinal cells, Vero, CV1, kidney (e.g., HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK), HeLa, HepG2, WI38, MRC 5, Colo205, HB 8065, HL-60, (e.g., BHK21), Jurkat, Daudi, A431 (epidermal), CV-1, U937, 3T3, L cells, C127 cells, SP2 / 0, NS-0, MMT 060562, Sertoli cells, BRL 3 A cells, HT1080 cells, myeloma cells, tumor cells, and cell lines derived from the foregoing cells. In some embodiments, the cell comprises one or more viral genes.

[0033] "Host cell protein(s)" or "HCP(s): As used herein, refers to proteins that may be present in a cell extract or preparation, e.g., because they are produced by or otherwise contained within or on the host cell in which a fusion polypeptide described herein (e.g., a phosphorylated or unphosphorylated fusion polypeptide) is produced, and that are not a fusion polypeptide. In some embodiments, the provided technology (e.g., the provided production methods, e.g., the provided purification methods) eliminates or reduces HCPs from a preparation(s) of a fusion polypeptide (e.g., from a preparation of a phosphorylated fusion polypeptide described herein). A "reduced HCP preparation" refers to a preparation that contains reduced HCPs, e.g., compared to the amount present before application of the relevant purification step (e.g., provided herein) and / or compared to that achieved via a different purification technology. In some embodiments, the provided technology achieves the production of a fusion polypeptide preparation (e.g., a preparation of a phosphorylated fusion polypeptide) in which no HCP is detectable, e.g., using an ELISA method. In some embodiments, removal of HCPs can be monitored or assessed during or after purification of, e.g., a fusion polypeptide described herein (e.g., a phosphorylated form thereof) from a host cell, which in some embodiments can be an engineered mammalian cell described herein (e.g., expressing the fusion polypeptide and the kinase that phosphorylates it at a ratio in the range of about 4:1 to 10:1, e.g., about 8:1).

[0034] "Improved," "increased," or "reduced": As used herein, these terms, or grammatically equivalent relative terms, refer to a value relative to a comparable reference measurement. For example, in some embodiments, an assessment achieved with an agent of interest may be "improved" compared to an assessment obtained with a comparable reference agent. Alternatively or additionally, in some embodiments, an assessment achieved in a subject or system of interest may be "improved" compared to that obtained in the same subject or system under different conditions (e.g., before and after an event such as administration of a drug of interest) or in a different comparable subject (e.g., in a different comparable subject or system than the subject or system of interest, in the presence of one or more indicators of a particular disease, disorder, or condition of interest, or prior to exposure to the condition or drug). In some embodiments, relative terms refer to a statistically relevant difference (e.g., a difference of sufficient generality and / or magnitude to achieve statistical relevance). One of skill in the art will recognize or be able to readily determine the degree and / or generality of difference necessary or sufficient to achieve such statistical significance in a given context.

[0035] In vitro: The term "in vitro," as used herein, refers to events that take place in an artificial environment, e.g., a test tube or reaction vessel, cell culture, etc., rather than within a multicellular organism.

[0036] In vivo: As used herein, refers to events that occur within multicellular organisms, such as humans and non-human animals. In the context of cell-based systems, the term is also used to refer to events that occur within living cells (e.g., rather than in vitro systems).

[0037] Isolated: As used herein, means a substance and / or entity that is (1) separated from at least some of the components with which it was associated when originally produced (whether in nature and / or in an experimental setting), and / or (2) artificially designed, produced, prepared, and / or manufactured. Isolated substances and / or entities may be separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% of other components with which they were originally associated. In some embodiments, an isolated agent is about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or greater than about 99% pure. As used herein, a substance is "pure" if it is substantially free of other components. In some embodiments, as will be understood by those skilled in the art, a substance may be considered "isolated" or even "pure" even after being combined with certain other components, such as, for example, one or more carriers or excipients (e.g., buffers, solvents, water, etc.). In such embodiments, the isolation or purity of a substance is calculated without including such carriers or excipients. For example, in some embodiments, a biological polymer, such as a polypeptide or polynucleotide, occurring in nature is considered "isolated" if: a) by its origin or source, it is not associated with some or all of the components that naturally accompany it in nature; b) it is substantially free of other polypeptides or nucleic acids of the same species from the species that produces it in nature; or c) it is expressed by or otherwise associated with components from cells or other expression systems other than the species that produces it in nature. Thus, for example, in some embodiments, a polypeptide that is chemically synthesized or synthesized in a cellular system different from that which produces it in nature is considered to be an "isolated" polypeptide.Alternatively or additionally, in some embodiments, a polypeptide that has been subjected to one or more purification techniques may be considered to be an "isolated" polypeptide so long as it is separated from a) other components with which it is associated in nature, and / or b) other components with which it was associated when originally produced.

[0038] Linker: As used herein, the term "linker" refers to a portion of a multi-element agent that connects different elements to one another. For example, one of skill in the art will understand that polypeptides whose structure includes two or more functional or organizational portions or domains often contain a stretch of amino acids between them that joins such portions or domains. In some embodiments, polypeptides containing linker elements have an overall structure of the general form S1-L-S2, where S1 and S2, which may be the same or different, represent two portions or domains associated with each other by the linker. In some embodiments, the polypeptide linker is at least 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, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more amino acids in length. In some embodiments, the linker is characterized by its tendency not to adopt a rigid three-dimensional structure, but rather to provide flexibility to the polypeptide. A variety of different linker elements that may be suitably used in engineering polypeptides (e.g., fusion polypeptides) are known in the art (see, e.g., Holliger et al., Proc. Natl. Acad. Sci. USA 90:6444, 1993; Poljak et al. Structure 2:1121, 1994).

[0039] Modulator: The term "modulator" is used to refer to an entity whose presence or level in a system in which an activity of interest is observed correlates with a change in the level and / or characteristics of that activity when compared to what is observed under otherwise equivalent conditions in the absence of the modulator. In some embodiments, a modulator is an activator, in that its activity is increased compared to what is observed under otherwise equivalent conditions in the absence of the modulator. In some embodiments, a modulator is an antagonist or inhibitor, in that its activity is reduced in its presence compared to what is observed under otherwise equivalent conditions in the absence of the modulator. In some embodiments, a modulator interacts directly with a target entity whose activity is of interest. In some embodiments, a modulator interacts indirectly with a target entity whose activity is of interest (i.e., directly through an intermediate agent that interacts with the target entity). In some embodiments, a modulator affects the level of a target entity of interest. Alternatively or additionally, in some embodiments, a modulator affects the activity of a target entity of interest without affecting the level of the target entity. In some embodiments, a modulator affects both the level and activity of a target entity of interest, such that the observed difference in activity is not entirely explained by or equal to the observed difference in level.

[0040] Moiety: Those skilled in the art will understand that a "moiety" is a defined chemical group or entity that has a particular structure and / or activity as described herein. Typically, a "moiety" is a portion of a molecule or entity that is smaller than the whole.

[0041] Variant: As used herein, the term "variant" refers to an entity that exhibits significant structural identity with a reference substance, but that differs structurally from the reference substance in the presence or level of one or more chemical moieties compared to the reference substance. In many embodiments, a variant also differs functionally from the reference substance. Generally, whether a particular entity is properly considered a "variant" of a reference substance is based on the degree of structural identity with the reference substance. As one skilled in the art will understand, any biological or chemical reference entity has certain characteristic structural elements. By definition, a variant is a different chemical entity that shares one or more such characteristic structural elements. To give a few examples, a small molecule may have a distinctive core structural element (e.g., a macrocyclic core) and / or one or more distinctive pendant moieties, such that variants of the small molecule share the core structural element and distinctive pendant moieties but differ in other pendant moieties and / or in the type of bond present within the core (single bond vs. double bond, E vs. Z, etc.); a polypeptide may have distinctive sequence elements comprised of multiple amino acids that have designated positions relative to each other in linear or three-dimensional space and / or that contribute to a particular biological function; and a nucleic acid may have distinctive sequence elements comprised of multiple nucleotide residues that have designated positions relative to each other in linear or three-dimensional space. For example, a variant polypeptide can differ from a reference polypeptide as a result of one or more differences in amino acid sequence and / or one or more differences in chemical moieties (e.g., carbohydrates, lipids, etc.) covalently attached to the polypeptide backbone. In some embodiments, the variant polypeptide exhibits an overall sequence identity with the reference polypeptide that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%. Alternatively or additionally, in some embodiments, the variant polypeptide does not share at least one characteristic sequence element with the reference polypeptide. In some embodiments, the reference polypeptide has one or more biological activities. In some embodiments, the variant polypeptide shares one or more of the biological activities of the reference polypeptide.In some embodiments, a variant polypeptide lacks one or more of the biological activities of a reference polypeptide, hi some embodiments, a variant polypeptide exhibits a reduced level of one or more biological activities compared to the reference polypeptide.

[0042] Operably linked: As used herein, refers to a juxtaposition wherein the described components are in a relationship permitting them to function in their intended manner. A control element "operably linked" to a functional element is associated such that expression and / or activity of the functional element is achieved under conditions compatible with the control element. In some embodiments, an "operably linked" control element is adjacent (e.g., covalently linked) to the coding element of interest, and in some embodiments, the control element acts in trans or remotely from the functional element of interest.

[0043] Patient: As used herein, the term "patient" refers to any organism to which provided compositions are or can be administered, e.g., for experimental, diagnostic, prophylactic, cosmetic, and / or therapeutic purposes. Typical patients include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and / or humans). In some embodiments, the patient is human. In some embodiments, the patient is suffering from or susceptible to one or more disorders or conditions. In some embodiments, the patient exhibits one or more symptoms of a disorder or condition. In some embodiments, the patient has been diagnosed with one or more disorders or conditions. In some embodiments, the disorder or condition is or includes cancer or the presence of one or more tumors. In some embodiments, the patient is undergoing or has undergone a particular therapy to diagnose and / or treat the disease, disorder, or condition.

[0044] Pharmaceutical composition: As used herein, the term "pharmaceutical composition" refers to an active agent formulated with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in a unit dosage suitable for administration in a treatment regimen that exhibits a statistically significant likelihood of achieving a predetermined therapeutic effect when administered to a relevant population. In some embodiments, pharmaceutical compositions may be specially formulated for administration in solid or liquid form, including, for example, those adapted for specific routes of administration as described herein.

[0045] Pharmaceutically acceptable: As used herein, the phrase "pharmaceutically acceptable" is used to refer to agents or entities that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and / or animals without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0046] Pharmaceutically acceptable carrier: As used herein, the term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, or vehicle encapsulating material, that is involved in the delivery or transport of a compound of interest from one organ or body part to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can function as pharmaceutically acceptable carriers include sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; excipients such as powdered tragacanth, malt, gelatin, talc, cocoa butter, and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar, buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, pH buffer solutions, polyesters, polycarbonates, and / or polyanhydrides, and other non-toxic compatible substances employed in pharmaceutical formulations.

[0047] Polypeptide: As used herein, refers to a polymeric chain of amino acids. In some embodiments, a polypeptide has a naturally occurring amino acid sequence. In some embodiments, a polypeptide has a non-naturally occurring amino acid sequence. In some embodiments, a polypeptide has an engineered amino acid sequence, in that it has been designed and / or generated through the act of man. In some embodiments, a polypeptide can comprise or consist of natural amino acids, non-natural amino acids, or both. In some embodiments, a polypeptide can comprise or consist of only natural amino acids or only non-natural amino acids. In some embodiments, a polypeptide can comprise D-amino acids, L-amino acids, or both. In some embodiments, a polypeptide can comprise only D-amino acids. In some embodiments, a polypeptide can comprise only L-amino acids. In some embodiments, a polypeptide can comprise one or more pendant groups or other modifications, e.g., modification of or attachment to one or more amino acid side chains, at the N-terminus of the polypeptide, at the C-terminus of the polypeptide, or any combination thereof. In some embodiments, such pendant groups or modifications can be selected from the group consisting of acetylation, amidation, lipidation, methylation, pegylation, etc. (including combinations thereof). In some embodiments, a polypeptide may be cyclic and / or include a cyclic portion. In some embodiments, a polypeptide is not cyclic and / or does not include a cyclic portion. In some embodiments, a polypeptide is linear. In some embodiments, a polypeptide may be or include a stapled polypeptide. In some embodiments, the term "polypeptide" may be appended to the name of a reference polypeptide, activity, or structure, and in such cases, it is used herein to refer to polypeptides that share a related activity or structure and can therefore be considered members of the same class or family of polypeptides. For each such class, the specification provides, and / or one of skill in the art will be aware of, exemplary polypeptides within the class whose amino acid sequence and / or function are known.In some embodiments, such exemplary polypeptides are reference polypeptides of a class or family of polypeptides. In some embodiments, members of a polypeptide class or family exhibit significant sequence homology or identity with the reference polypeptide of the class (and in some embodiments, with all polypeptides in the class), share common sequence motifs (e.g., characteristic sequence elements), and / or share a common activity (in some embodiments, at a similar level or within a specified range). For example, in some embodiments, member polypeptides exhibit an overall degree of sequence homology or identity with the reference polypeptide that is at least about 30-40%, and often greater than about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, and / or contain at least one region (e.g., a conserved region that, in some embodiments, is or may include a characteristic sequence element) that exhibits very high sequence identity, often greater than 90%, or even 95%, 96%, 97%, 98%, or 99%. Such conserved regions typically encompass at least 3-4, and often up to 20 or more amino acids; in some embodiments, the conserved region encompasses at least one stretch of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more contiguous amino acids. In some embodiments, a related polypeptide may comprise or consist of a fragment of a parent polypeptide. In some embodiments, a useful polypeptide may comprise or consist of multiple fragments, each of which is found in the same parent polypeptide in a different spatial arrangement relative to each other than that found in the polypeptide of interest (e.g., a fragment directly linked to the parent may be spatially separated in the polypeptide of interest, or vice versa, and / or the fragments may be present in a different order in the polypeptide of interest than in the parent), and thus the polypeptide of interest is a derivative of that parent polypeptide.

[0048] Predetermined: Predetermined means deliberately selected, as opposed to, for example, occurring or achieving randomly.

[0049] Pure: As used herein, an agent or entity is "pure" if it is substantially free of other components. For example, a preparation containing greater than about 90% of a particular agent or entity is typically considered a pure preparation. In some embodiments, the agent or entity is at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% pure.

[0050] Recombinant: as used herein is intended to mean a polypeptide that is designed, engineered, prepared, expressed, made, manufactured, and / or isolated by recombinant means, e.g., a polypeptide expressed using a recombinant expression vector transfected into a host cell; a polypeptide isolated from a recombinant combinatorial human polypeptide library; a polypeptide isolated from an animal (e.g., mouse, rabbit, sheep, fish, etc.) that has been transgenic for or otherwise engineered to express a gene(s) or genetic component(s) that encodes and / or directs the expression of a polypeptide or one or more component(s), portion(s), element(s), or domain(s) thereof; and / or a polypeptide prepared, expressed, made, or isolated by any other means, including splicing or ligation of selected nucleic acid sequence elements together, chemical synthesis of selected sequence elements, and / or alternative generation of a nucleic acid that encodes and / or directs the expression of a polypeptide or one or more component(s), portion(s), element(s), or domain(s) thereof. In some embodiments, one or more of such selected sequence elements are found in nature. In some embodiments, one or more of such selected sequence elements are designed in silico. In some embodiments, one or more of such selected sequence elements arise from mutagenesis (e.g., in vivo or in vitro) of known sequence elements, e.g., from natural or synthetic sources, e.g., in the germline of a source organism of interest (e.g., human, mouse, etc.).

[0051] Reference standard: As used herein, refers to a standard or control against which a comparison is made. For example, in some embodiments, an agent, animal, individual, population, sample, sequence, or value of interest is compared to a reference or control agent, animal, individual, population, sample, sequence, or value. In some embodiments, the reference or control is tested and / or determined substantially simultaneously with the test or determination of interest. In some embodiments, the reference or control is a historical reference or control, optionally embodied in a tangible medium. Typically, as understood by those skilled in the art, a reference or control is determined or characterized under conditions or circumstances equivalent to those being evaluated. Those skilled in the art will understand when sufficient similarity exists to justify reliance on and / or comparison to a particular potential reference or control.

[0052] Specific binding: As used herein, the term "specific binding" refers to the ability to distinguish between potential binding partners in an environment in which binding is to occur. A binding agent that interacts with one specific target in the presence of other potential targets is said to "specifically bind" to that interacting target. In some embodiments, specific binding is assessed by detecting or measuring the degree of association between the binding agent and its partner; in some embodiments, specific binding is assessed by detecting or measuring the degree of dissociation of the binding agent-partner complex; in some embodiments, specific binding is assessed by detecting or measuring the ability of a binding agent to compete with an alternative interaction of its partner with another entity. In some embodiments, specific binding is assessed by performing such detection or measurement over a range of concentrations.

[0053] Specific: The term "specific," as used herein with respect to an active agent, is understood by those skilled in the art to mean that the agent discriminates between potential target entities or aspects. For example, in some embodiments, an agent is said to bind "specifically" to its target if it preferentially binds to its target in the presence of one or more competing alternative targets. In many embodiments, specific interaction depends on the presence of particular structural features of the target entity (e.g., epitopes, clefts, binding sites). It should be understood that specificity need not be absolute. In some embodiments, specificity can be assessed relative to the specificity of a binding agent for one or more other potential target entities (e.g., competitors). In some embodiments, specificity is assessed relative to that of a reference specific binding agent. In some embodiments, specificity is assessed relative to that of a reference nonspecific binding agent. In some embodiments, an agent or entity does not detectably bind to competing alternative targets under conditions in which it binds to its own target entity. In some embodiments, a binding agent binds to its target entity with a higher on rate, a lower off rate, increased affinity, decreased dissociation, and / or increased stability when compared to competing alternative target(s).

[0054] Specificity: As known in the art, "specificity" is the degree of ability of a particular ligand to distinguish its own binding partner from other potential binding partners.

[0055] Subject: As used herein, the term "subject" refers to an organism, typically a mammal (e.g., a human, including in some embodiments prenatal human forms). In some embodiments, the subject is suffering from the relevant disease, disorder, or condition. In some embodiments, the subject is susceptible to the disease, disorder, or condition. In some embodiments, the subject exhibits one or more symptoms or characteristics of the disease, disorder, or condition. In some embodiments, the subject does not exhibit any symptoms or characteristics of the disease, disorder, or condition. In some embodiments, the subject is one who possesses one or more features characteristic of susceptibility to or risk for the disease, disorder, or condition. In some embodiments, the subject is a patient. In some embodiments, the subject is an individual to whom and / or to whom a diagnosis and / or treatment is administered and / or has been administered.

[0056] Therapeutic Agent: As used herein, the phrase "therapeutic agent" refers to any agent that has a therapeutic effect and induces a desired biological and / or pharmacological effect when administered to a subject. In some embodiments, a therapeutic agent is any substance that can be used to alleviate, ameliorate, relieve, inhibit, prevent, delay the onset of, reduce the severity of, and / or reduce the incidence of one or more symptoms or characteristics of a disease, disorder, and / or condition.

[0057] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" refers to an amount of a substance (e.g., a therapeutic agent, composition, and / or formulation) that, when administered as part of a treatment regimen, elicits a desired biological response. In some embodiments, a therapeutically effective amount of a substance is an amount sufficient to treat, diagnose, prevent, and / or delay the onset of a disease, disorder, and / or condition when administered to a subject suffering from or susceptible to the disease, disorder, and / or condition. As will be appreciated by those skilled in the art, the effective amount of a substance can vary depending on factors such as the desired biological endpoint, the substance being delivered, the target cell or tissue, and the like. For example, an effective amount of a compound in a formulation for treating a disease, disorder, and / or condition is an amount that relieves, ameliorates, alleviates, inhibits, prevents, delays the onset of, reduces the severity of, and / or reduces the frequency of occurrence of one or more symptoms or characteristics of the disease, disorder, and / or condition. In some embodiments, a therapeutically effective amount is administered in a single dose; in some embodiments, multiple unit doses are required to deliver a therapeutically effective amount.

[0058] Treatment: As used herein, the term "treatment" (also "treat" or "treating") refers to the administration of a therapy that partially or completely alleviates, improves, relieves, inhibits, delays the onset of, reduces the severity of, and / or reduces the incidence of one or more symptoms, characteristics, and / or causes of a particular disease, disorder, and / or condition. In some embodiments, such treatment may be treatment of a subject who does not exhibit symptoms of the associated disease, disorder, and / or condition and / or who exhibits only early signs of the disease, disorder, and / or condition. Alternatively or additionally, such treatment may be treatment of a subject who exhibits one or more established symptoms of the associated disease, disorder, and / or condition. In some embodiments, therapy may be treatment of a subject who has been diagnosed with the associated disease, disorder, and / or condition. In some embodiments, therapy may be treatment of a subject who is known to have one or more susceptibility factors that statistically correlate with an increased risk of developing the associated disease, disorder, and / or condition. Thus, in some embodiments, treatment may be prophylactic. In some embodiments, the treatment may be therapeutic.

[0059] Tumor: As used herein, the term "tumor" refers to an abnormal growth of cells or tissue. In some embodiments, a tumor may comprise precancerous (e.g., benign), malignant, premetastatic, metastatic, and / or non-metastatic cells. In some embodiments, a tumor is associated with or is a sign of cancer. In some embodiments, a tumor may be a dispersed tumor or a liquid tumor. In some embodiments, a tumor may be a solid tumor.

[0060] Variant: As used herein in the context of a molecule, e.g., a nucleic acid, protein, or small molecule, the term "variant" refers to a molecule that exhibits significant structural identity with a reference molecule but differs structurally from the reference molecule, e.g., in the presence or absence or level of one or more chemical moieties compared to the reference entity. In some embodiments, a variant also differs functionally from its reference molecule. Generally, whether a particular molecule is properly considered a "variant" of a reference molecule is based on the degree of structural identity with the reference molecule. As will be understood by those skilled in the art, any biological or chemical reference molecule will have certain characteristic structural elements. A variant, by definition, is a distinct molecule that shares one or more such characteristic structural elements but differs in at least one aspect from the reference molecule. To give some examples, a polypeptide may have characteristic sequence elements composed of multiple amino acids that have designated positions relative to each other in linear or three-dimensional space and / or contribute to a particular structural motif and / or biological function, and a nucleic acid may have characteristic sequence elements composed of multiple nucleotide residues that have designated positions relative to each other in linear or three-dimensional space. In some embodiments, a variant polypeptide or nucleic acid may differ from a reference polypeptide or nucleic acid as a result of one or more differences in amino acid or nucleotide sequence and / or one or more differences in chemical moieties (e.g., carbohydrates, lipids, phosphate groups) that are covalently attached components of the polypeptide or nucleic acid (e.g., to which the polypeptide or nucleic acid backbone is attached). In some embodiments, a variant polypeptide or nucleic acid exhibits an overall sequence identity with the reference polypeptide or nucleic acid that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%. In some embodiments, a variant polypeptide or nucleic acid does not share at least one characteristic sequence element with the reference polypeptide or nucleic acid. In some embodiments, a reference polypeptide or nucleic acid has one or more biological activities.In some embodiments, a variant polypeptide or nucleic acid shares one or more biological activities of a reference polypeptide or nucleic acid. In some embodiments, a variant polypeptide or nucleic acid lacks one or more biological activities of the reference polypeptide or nucleic acid. In some embodiments, a variant polypeptide or nucleic acid exhibits a reduced level of one or more biological activities compared to a reference polypeptide or nucleic acid. In some embodiments, a polypeptide or nucleic acid of interest is considered a "variant" of a reference polypeptide or nucleic acid if it has an amino acid or nucleotide sequence identical to that of the reference, but with minor sequence modifications at specific positions. Typically, less than about 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, or 2% of the residues in the variant are substituted, inserted, or deleted compared to the reference. In some embodiments, a variant polypeptide or nucleic acid contains about 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 substituted residue compared to the reference. In many cases, a variant polypeptide or nucleic acid contains very few (e.g., less than about 5, about 4, about 3, about 2, or about 1) substituted, inserted, or deleted functional residues (i.e., residues involved in a particular biological activity) compared to the reference. In some embodiments, a variant polypeptide or nucleic acid contains no more than about 5, about 4, about 3, about 2, or about 1 additions or deletions compared to the reference, and in some embodiments, no additions or deletions. In some embodiments, a variant polypeptide or nucleic acid contains less than about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 10, about 9, about 8, about 7, about 6, and typically less than about 5, about 4, about 3, or about 2 additions or deletions compared to the reference. In some embodiments, the reference polypeptide or nucleic acid is one found in nature. In some embodiments, the reference polypeptide or nucleic acid is a human polypeptide or nucleic acid.

[0061] Vector: As used herein, refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, into which additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0062] Virus inactivation or removal: As used herein, the term "virus inactivation or removal" refers to the inactivation or removal of viruses that may be contained in a sample, such as, for example, a cell extract or a fusion polypeptide preparation. In some embodiments, the viruses present in the sample may originate from the source material (e.g., host cells); alternatively or additionally, in some embodiments, the viruses present in the sample may have been introduced, for example, during processing of such a source material. Those skilled in the art will be aware of various techniques for achieving virus inactivation or removal, such as, for example, pH inactivation, chemical inactivation (e.g., through the use of chemical agents, e.g., detergents). Those skilled in the art will understand that "pH virus inactivation" involves (includes) exposing a virus (e.g., a sample containing a virus) to a pH that inactivates (e.g., has been established to inactivate) the virus.

[0063] Wild-type: As used herein, the term "wild-type" has its art-understood meaning and refers to a form of an entity (e.g., a polypeptide or nucleic acid) having a structure and / or activity found in nature in a "normal" (as opposed to mutated, diseased, or altered) state or situation. In some embodiments, more than one "wild-type" form of a particular polypeptide or nucleic acid may exist in nature, for example, as "alleles" of a particular gene or normal variants of a particular polypeptide. In some embodiments, the form(s) of a particular polypeptide or nucleic acid that are most commonly observed in a population (e.g., the human population) are the "wild-type" forms.

[0064] The present disclosure particularly provides methods of preparation that include purifying a phosphorylated form of a fusion polypeptide (e.g., as described in published international patent application WO 2020 / 263399). In particular, the present disclosure relates to the preparation of a phosphorylated form of a fusion polypeptide that includes: (a) an immunomodulatory polypeptide that includes an immune agonist portion; and (b) a metal hydroxide-binding polypeptide, the amino acid sequence of which includes multiple phosphorylation sites such that the metal hydroxide-binding polypeptide can adopt phosphorylated and unphosphorylated forms.

[0065] Fusion Polypeptides In some embodiments, fusion polypeptides according to the present disclosure may adopt phosphorylated and unphosphorylated forms.

[0066] Immunomodulatory Polypeptides In some embodiments, a fusion polypeptide of the present disclosure comprises at least one immunomodulatory polypeptide.

[0067] In some embodiments, a fusion polypeptide comprises two or more immunomodulatory polypeptides (e.g., two or more immune agonist moieties). In some such embodiments, a fusion polypeptide comprises two or more of the same immunomodulatory polypeptides, and in some such embodiments, all of the immunomodulatory polypeptides within a fusion polypeptide according to the present disclosure are the same. In some embodiments, a fusion polypeptide comprises two or more immunomodulatory polypeptides that are different from each other.

[0068] In some embodiments, the immunomodulatory polypeptide is or comprises at least one immune agonist moiety. In some embodiments, the immune agonist moiety is or comprises a functional fragment of a parent (e.g., wild-type) polypeptide, e.g., in some embodiments, the immunomodulatory polypeptide is or comprises a functional fragment that is a signal transduction-capable fragment. In some embodiments, the immunomodulatory polypeptide comprises one, two, three, four, five, or six immune agonist moieties.

[0069] Thus, in some embodiments, an immunomodulatory polypeptide may comprise two or more immune agonist moieties, which in various embodiments may be the same or different. In some such embodiments, two or more such immune agonist moieties are the same, and in some embodiments, all such immune agonist moieties are the same. In some embodiments, an immunomodulatory polypeptide comprises two or more immune agonist moieties that are different from one another, and in some embodiments, no two such immune agonist moieties are the same.

[0070] In some embodiments, the fusion polypeptide comprises two or more immunomodulatory polypeptides (e.g., two or more immunoagonist moieties) that comprise at least two subtypes of immunomodulatory polypeptides (e.g., immunoagonist moieties), e.g., such that the fusion polypeptide comprises at least two first subtypes and at least two second subtypes.

[0071] In some embodiments, the immunomodulatory polypeptide (eg, immune agonist moiety) activates or inhibits the activity of (eg, is capable of signal transduction) cells of the immune system.

[0072] For example, in some embodiments, signaling capability is characterized by the immune agonist portion(s) or functional fragments thereof exhibiting equivalent binding to a reference standard (e.g., a wild-type polypeptide) when assessed for binding to a specific binding partner. For example, in some embodiments, signaling capability is characterized by the immune agonist portion(s) or functional fragments thereof exhibiting equivalent biological effect as a reference standard (e.g., a wild-type polypeptide) when assessed for said biological effect, e.g., in vitro or in vivo.

[0073] For example, in some embodiments, the immunomodulatory polypeptide (e.g., immune agonist moiety) is an immune response stimulating moiety, such as, but not limited to, a cytokine, a chemokine, an agonist antibody, an immune checkpoint inhibitor, or a combination thereof.

[0074] In some embodiments, the immunomodulatory polypeptide promotes the differentiation and proliferation of immune cells (e.g., T cells). In some embodiments, the immunomodulatory polypeptide enhances the production of interferon gamma.

[0075] In some embodiments, the immunomodulatory polypeptide comprises an interleukin-12 (IL-12) immunomodulatory polypeptide (eg, an IL-12 immunoagonist moiety).

[0076] IL-12 is a proinflammatory cytokine that plays an important role in innate and adaptive immunity. Wild-type IL-12 is a heterodimeric protein containing two subunits, p35 (IL-12A; GenBank GeneID: 3592) and p40 (IL-12B; GenBank GeneID: 3593), connected by disulfide bonds. Binding of IL-12 to the IL-12 receptor complex (IL-12Rβ1 / IL-12Rβ2) on T cells and natural killer (NK) cells leads to signal transduction via signal transducer and activator of transcription 4 (STAT4) and subsequent interferon-γ (IFN-γ) production and secretion.

[0077] The IL-12 subunits IL-12A and IL-12B can also form heterodimers with other IL-12 family members. For example, IL-12A can also dimerize with Epstein-Barr virus-induced gene 3 (EBI3) to form the IL-12 family member, and IL-35 and IL-12B can dimerize with p19 monomers to form the IL-12 family member IL-23.

[0078] IL-12 plays an important role in innate and adaptive immune responses, and dysregulation of IL-12 is involved in several disease states. Exemplary disease states include, but are not limited to, inflammatory bowel disease, psoriasis, diabetes mellitus, multiple sclerosis, rheumatoid arthritis, cancer, lupus erythematosus, primary biliary cholangitis, and Sjögren's syndrome (Ullrich et al. EXCLI journal vol.19 1563-1589.11 Dec.2020). The use of IL-12 as a therapeutic tool has been extensively studied, including in the treatment of tumors (Nastala CL et al. J Immunol.1994 Aug 15; Lasek et al. Cancer immunology, immunotherapy: CII vol.63,5(2014):419-35).

[0079] In some embodiments, the immunomodulatory polypeptides disclosed herein are or comprise an IL-12 immune agonist moiety. In some embodiments, the immunomodulatory polypeptides disclosed herein comprise multiple IL-12 immune agonist moieties. In some embodiments, the immunomodulatory polypeptides disclosed herein comprise exactly two IL-12 immune agonist moieties. In some embodiments, two or more IL-12 immune agonist moieties of a multiple (e.g., two) IL-12 immune agonist moieties are the same moiety. In some such embodiments, the multiple (e.g., two) IL-12 immune agonist moieties are different moieties. In some such embodiments, the IL-12 immune agonist moiety comprises an IL-12A polypeptide or a functional fragment thereof. In some embodiments, the IL-12 immune agonist moiety comprises an IL-12B polypeptide or a functional fragment thereof.

[0080] In some embodiments, the immunomodulatory polypeptides disclosed herein comprise an IL-12 immune agonist portion comprising an IL-12A polypeptide or a functional fragment thereof and an IL-12 immune agonist portion comprising an IL-12B polypeptide or a functional fragment thereof.

[0081] In some embodiments, the IL-12B immune agonist moiety is located N-terminal to the IL-12A immune agonist moiety in the immune modulatory polypeptide. In some embodiments, the IL-12A immune agonist moiety is located N-terminal to the IL-12B immune agonist moiety in the immune modulatory polypeptide.

[0082] In some embodiments, immunomodulatory polypeptides comprising multiple (e.g., two) IL-12 immune agonist moieties (e.g., IL-12A and / or IL-12B) are directly linked. In some embodiments, immunomodulatory polypeptides comprising multiple (e.g., two) IL-12 moieties (e.g., IL-12A and / or IL-12B) are linked via a first linker. Non-limiting examples of linkers are discussed elsewhere herein.

[0083] In some embodiments, the immunomodulatory polypeptides disclosed herein comprise an IL-12A and / or IL-12B immune agonist moiety that comprises a variant. In some embodiments, the variant of the IL-12A and / or IL-12B immune agonist moiety comprises a substitution, deletion, addition, and / or insertion compared to the wild-type IL-12A or IL-12B polynucleotide or amino acid sequence. In some embodiments, the IL-12A and / or IL-12B immune agonist moiety comprises a plurality of variants. In some embodiments, the plurality of variants comprises one or more substitutions, deletions, additions, and / or insertions compared to the wild-type IL-12A or IL-12B. In some embodiments, the variants comprise substitutions that do not change the amino acid sequence compared to the wild-type IL-12A or IL-12B. In some embodiments, the IL12 variants comprise mutations.

[0084] In some embodiments, the immunomodulatory polypeptides disclosed herein comprise IL-12A and / or IL-12B immune agonist portions that are functional fragments thereof (e.g., fragments capable of signal transduction). In some embodiments, the immunomodulatory polypeptide comprises a functional IL-12A fragment. In some embodiments, the immunomodulatory polypeptide comprises a functional IL-12B fragment. In some embodiments, the immunomodulatory polypeptide comprises full-length IL-12A and a functional IL-12B fragment. In some embodiments, the immunomodulatory polypeptide comprises full-length IL-12B and a functional IL-12A fragment.

[0085] In some embodiments, an IL-12A or IL-12B fragment comprises or consists of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more of the monomeric units (e.g., residues) found in wild-type IL-12A or IL-12B.

[0086] In some embodiments, the immunomodulatory polypeptides disclosed herein comprise an IL-12A and / or IL-12B immunoagonist moiety that is a human IL-12A and / or IL-12B immunoagonist moiety.

[0087] In some embodiments, the immunomodulatory polypeptides disclosed herein comprise an IL-12B immunoagonist portion having at least 80% sequence identity to SEQ ID NO: 3, e.g., at least 85%, such as at least 90%, for example, at least 91%, such as at least 92%, for example, at least 93%, such as at least 94%, for example, at least 95%, for example, at least 96%, such as at least 97%, for example, at least 98%, for example, at least 99% sequence identity to SEQ ID NO: 3. In some embodiments, the immunomodulatory polypeptides disclosed herein comprise an IL-12A immunoagonist portion having at least 80% sequence identity to SEQ ID NO: 4, e.g., at least 85%, such as at least 90%, for example, at least 91%, such as at least 92%, for example, at least 93%, such as at least 94%, for example, at least 95%, for example, at least 96%, such as at least 97%, for example, at least 98%, for example, at least 99% sequence identity to SEQ ID NO: 4. In some embodiments, the immunomodulatory polypeptides disclosed herein comprise an IL-12B immune agonist portion having at least 80% sequence identity to SEQ ID NO:3 and an IL-12A immune agonist portion having at least 80% sequence identity to SEQ ID NO:4.

[0088] In some embodiments, the immunomodulatory polypeptide disclosed herein comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO:5, such as at least 85%, for example, at least 90%, for example, at least 91%, for example, at least 92%, for example, at least 93%, for example, at least 94%, for example, at least 95%, for example, at least 96%, for example, at least 97%, for example, at least 98%, for example, at least 99% sequence identity to SEQ ID NO:5.

[0089] Without being bound by any particular theory, hydroxyl substitutions (e.g., at phosphate groups) may increase the adsorption of polypeptides via ligand exchange with metal hydroxides (e.g., aluminum hydroxide) and may further improve the tumor retention and antitumor efficacy of such polypeptides (e.g., particularly immunomodulatory polypeptides and fusion polypeptides comprising metal hydroxide-binding polypeptides in which such hydroxyl substitutions have occurred).

[0090] In some embodiments, immunomodulatory polypeptides according to the present invention can adopt phosphorylated and unphosphorylated forms. In some embodiments, immunomodulatory polypeptides comprise at least one amino acid that can be phosphorylated. In some embodiments, immunomodulatory polypeptides comprise at least one kinase target motif. In some embodiments, immunomodulatory polypeptides do not comprise a kinase target motif. In these embodiments, immunomodulatory polypeptides still comprise amino acids that can be phosphorylated. In some embodiments, immunomodulatory polypeptides comprising one or more phosphorylated amino acids contribute to strong binding of metals to metal hydroxides, e.g., aluminum hydroxide. Table 1 lists exemplary serine residues that can be phosphorylated in immunomodulatory domains (e.g., S43, S154, S168, S233, S365, S398, S481 of SEQ ID NO: 2). In some embodiments, immunomodulatory polypeptides comprise at least one phosphorylated serine. In some embodiments, immunomodulatory polypeptides comprise at least two phosphorylated serine residues. In some embodiments, immunomodulatory polypeptides comprise at least three phosphorylated serine residues. In some embodiments, immunomodulatory polypeptides comprise at least four phosphorylated serine residues. In some embodiments, the immunomodulatory polypeptide comprises at least 5 phosphorylated serine residues, e.g., 6 serine residues, e.g., 7 serine residues, e.g., 8 serine residues, e.g., 9 serine residues, e.g., 10 serine residues.

[0091] Metal hydroxide-binding peptides In some embodiments, a fusion polypeptide of the present disclosure comprises at least one metal-binding polypeptide.

[0092] The present disclosure provides metal hydroxide-binding polypeptides, and fusion polypeptides comprising them, that demonstrate high levels of adsorption to metal hydroxides and further desirable manufacturing characteristics (e.g., one or more of reproducibility, consistency, production of homogeneously phosphorylated fusion polypeptide preparations, etc.).

[0093] In some embodiments, a fusion polypeptide comprises two or more metal-binding polypeptides (e.g., two or more alum-binding polypeptides). In some embodiments, a fusion polypeptide comprises two or more metal-binding polypeptides that are the same; in some such embodiments, all metal-binding polypeptides in a fusion polypeptide according to the present disclosure are the same. In some such embodiments, a fusion polypeptide comprises two or more metal-binding polypeptides that are different from each other.

[0094] As discussed above, metal-binding peptides can be fused to immunomodulatory polypeptides, allowing them to bind strongly to metal hydroxides, such as aluminum hydroxide. Various immunomodulatory polypeptides can be fused to metal-binding peptides. Without being bound by theory, metal-binding polypeptides adsorbed to alum in serum can be used to retain proteins and peptides in tumors.

[0095] In some embodiments, the metal hydroxide-binding polypeptide comprises an amino acid sequence containing multiple phosphorylation sites, allowing it to assume phosphorylated and unphosphorylated forms. In some embodiments, the metal hydroxide-binding polypeptide comprises at least one kinase target motif. The target kinase motif comprises an amino acid that is phosphorylated by a kinase. Amino acids that are typically phosphorylated contain hydroxyl groups, such as serine (Ser, S) residues, threonine (Thr, T) residues, and tyrosine (Tyr, Y) residues. A kinase motif refers to the adjacent amino acid sequence at the N-terminus and / or C-terminus of an amino acid residue that can be phosphorylated. Without wishing to be bound by any theory, many kinases contain structural features that confer specificity, such that the kinase phosphorylates a particular amino acid (e.g., serine, threonine, or tyrosine) in a particular kinase target motif.

[0096] The kinase target motifs recognized vary greatly depending on the specific type of kinase. In some embodiments, the present disclosure provides metal hydroxide-binding polypeptides that include one or more kinase target motifs of secretory pathway kinases. The secretory pathway is the pathway by which cells secrete proteins and / or other biomolecules into the extracellular space and refers to the endoplasmic reticulum (ER), Golgi apparatus (Golgi), plasma membrane, and lysosomal storage compartments, as well as the vesicles that travel between them. Secretory pathway kinases are localized throughout the secretory pathway (e.g., in the ER, Golgi, etc.) and function to phosphorylate proteins destined for secretion (Sreelatha et al. Biochimica et biophysica acta vol. 1854, 10 Pt B (2015): 1687-93).

[0097] In some embodiments, the relevant kinase is a naturally occurring secretory pathway kinase (e.g., endogenously targeted to and functioning in the secretory pathway). In some embodiments, the secretory pathway kinase comprises a signal sequence that targets the kinase to the secretory pathway. Naturally occurring human secretory pathway kinases include, for example, four-jointed box kinase 1, Fam20A, Fam20B, Fam20C, vertebrate lonesome kinase (VLK), SGK196, and Fam69A, Fam69B, and Fam69C.

[0098] In some embodiments, the relevant kinase is a non-naturally occurring secretory pathway kinase, hi some embodiments, the non-naturally occurring kinase is produced by linking a secretory signal peptide to a kinase that is endogenously localized in a cellular compartment of the non-secretory pathway.

[0099] In some embodiments, the kinase target motif is a target kinase motif of a secretory pathway kinase. In some embodiments, the target kinase motif of a secretory pathway kinase comprises an SXE motif. For example, Fam20C has been shown to phosphorylate serine and kinase target motifs containing the amino acid sequences Ser-X-Glu (e.g., SXE), Ser-X-pSer (e.g., SX-pS), and Ser-X-Gln-XX-Asp-Glu-Glu (SXQXXDEE), where X is any amino acid and pS is phosphorylated serine (Mercier, et al (1981) Biochimie, 63:1-17; Mercier et al (1971) Eur J. Biochem. 23:41-51; Lasa-Benito (1996) FEES Lett. 382:149; Brunati, et al (2000) 3:765, Tagliabraccci, et al (2015) Cell 161:1619-1632; Tagliabraccci, et al (2015) Cell 161:1619-1632). al (2012) Science 336:1150-1153). In some embodiments, the target kinase motif comprises the amino acid sequence SEEE. In some embodiments, the target kinase motif comprises the amino acid sequence SEEA. In some embodiments, the target kinase motif comprises the amino acid sequence SEEQ. In some embodiments, the target kinase motif comprises the amino acid sequence SEE.

[0100] In some embodiments, the metal hydroxide-binding polypeptide comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 target kinase motifs. In some embodiments, the metal hydroxide-binding polypeptide comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 SXE motifs. In some embodiments, the metal hydroxide-binding polypeptide comprises four or more SXE motifs. In some embodiments, the metal hydroxide-binding polypeptide comprises eight SXE motifs. In some embodiments, the number of target kinase motifs (e.g., SXE motifs) contributes to the number of phosphorylated residues on the metal hydroxide-binding polypeptide. In some embodiments, the metal hydroxide-binding polypeptide comprises eight SEE motifs.

[0101] In some embodiments, the metal hydroxide-binding polypeptide is a metal hydroxide-binding polypeptide whose amino acid sequence comprises multiple phosphorylation sites. In some embodiments, the multiple phosphorylation sites comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 target kinase motifs. In some embodiments, the multiple phosphorylation sites comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 SXE motifs. In some embodiments, the multiple phosphorylation sites comprise five or more SXE motifs. In some embodiments, the multiple phosphorylation sites comprise nine or more SXE motifs. In some embodiments, the number of target kinase motifs (e.g., SXE motifs) contributes to the number of phosphorylated residues on the metal hydroxide-binding polypeptide.

[0102] In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 target kinase motifs (e.g., SXE motifs) are immediately adjacent to (e.g., linked to) the next target kinase (e.g., SXE motif). In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 target kinase motifs (e.g., SXE motifs) are separated from (e.g., linked to) the next target kinase motif (e.g., SXE motif) by a spacer. In some embodiments, the spacer comprises at least one glycine residue. In some embodiments, the spacer comprises multiple glycine residues. In some embodiments, the spacer comprises three glycine residues. In some embodiments, the spacer comprises at least four glycine residues. In some embodiments, the spacer has a sequence comprising four glycine residues. In some embodiments, the spacer has an amino acid sequence comprising GGGSGGGG. In some embodiments, the spacer has an amino acid sequence that includes GGGEGGGG. In some embodiments, the spacer has an amino acid sequence that includes GGGGG. In some embodiments, the spacer has an amino acid sequence that includes GGGG.

[0103] In some embodiments, the metal hydroxide-binding polypeptide comprises four SXE motifs and three spacers comprising four glycine residues. In some embodiments, the metal hydroxide-binding polypeptide comprises six SXE motifs and five spacers comprising four glycine residues. In some embodiments, the metal hydroxide-binding polypeptide comprises eight SXE motifs and seven spacers comprising four glycine residues. In some embodiments, the metal hydroxide-binding polypeptide comprises eight SXE motifs and eight spacers comprising four glycine residues. In some embodiments, the metal hydroxide-binding polypeptide comprises eight motifs having the amino acid sequence SEE and eight spacers comprising four glycine residues.

[0104] In some embodiments, the metal hydroxide-binding polypeptide comprises a terminal sequence (e.g., an amino acid sequence) at the C-terminus of the fusion polypeptide. In some embodiments, the terminal sequence comprises multiple amino acid residues. In some embodiments, the multiple amino acid residues comprise GGGG. In some such embodiments, the terminal sequence comprises the amino acid sequence GGGGS.

[0105] In some embodiments, the desired (e.g., optimal) number of kinase target motifs and / or spacing of kinase motifs can be determined based on, for example, one or more of: a desired phosphate content that achieves strong metal hydroxide retention and / or avoidance of one or more manufacturing challenges (e.g., as recognized by the present disclosure and that may be associated with highly phosphorylated elements). In some embodiments, the desired (e.g., optimal) number of kinase motifs and / or spacing of kinase motifs allows exposure of the polypeptide to a kinase to achieve a desired level of fusion polypeptide phosphorylation. In some embodiments, improved fusion polypeptides as described herein result in one or more of improved reproducibility, consistency, and / or production of homogeneously phosphorylated fusion polypeptides. For example, in some embodiments, the provided technology achieves reproducible production of equivalent preparations (e.g., preparations that are consistent within established parameters) of fusion polypeptides (e.g., phosphorylated fusion polypeptides) and / or complexes as described herein. For example, in some embodiments, the provided technology achieves reduced immunogenicity compared to an appropriate reference standard.

[0106] In some embodiments, the degree of phosphorylation (e.g., average number of phosphate molecules per polypeptide) is 0.5-7, 1-7, 2-7, 3-7, 4-7, 5-7, 6-7, 0.5-6, 0.5-5, 0.5-4, 1-6, 2-6, 3-6, 4-6, 5-6, 7-8, 8-9, 9-10, 10-11, 11-12, or 13-14. In some embodiments, the degree of phosphorylation (e.g., average number of phosphate molecules per polypeptide) is 3, 3.5, 4.0, 4.5, 5.0, 5.5, 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, 7.10, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.5, 11, 11.5, or 12.

[0107] Linker In some embodiments, the fusion polypeptides described herein may include one or more linkers and / or spacers.

[0108] For example, in some embodiments, a fusion polypeptide comprises an immunomodulatory polypeptide comprising a first and a second immune agonist moiety, in some embodiments, the first immune agonist moiety and the second immune agonist moiety are linked via a first linker.

[0109] In some embodiments, a fusion polypeptide of the present disclosure comprises an immunomodulatory polypeptide and a metal hydroxide-binding polypeptide, hi some embodiments, the immunomodulatory polypeptide and the metal hydroxide-binding polypeptide are linked via a second linker.

[0110] In some embodiments, the first linker and / or the second linker is a polypeptide linker. In some embodiments, the polypeptide linker is synthetic. For example, a synthetic polypeptide linker can include a non-naturally occurring polypeptide that is a modified form of a naturally occurring polypeptide.

[0111] In some embodiments, polypeptide linkers of the present disclosure are at least one amino acid in length and can be any suitable number of amino acids, hi some embodiments, polypeptide linkers are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 amino acids in length.

[0112] In some embodiments, the first linker comprises a polypeptide linker. In some embodiments, the first linker comprises or consists of a glycine-serine (Gly-Ser or GS linker). A Gly-Ser linker is a polypeptide linker consisting of glycine and serine residues. In some embodiments, a Gly-Ser linker is (Gly4Ser) nwherein n is a positive integer (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some embodiments, the Gly-Ser linker is (Gly4Ser)1. In some embodiments, the Gly-Ser linker is (Gly4Ser)2. In some embodiments, the Gly-Ser linker is (Gly4Ser)3. In some embodiments, the Gly-Ser linker is (Gly4Ser)4. In some embodiments, the Gly-Ser linker is (Gly4Ser)5. In some embodiments, the Gly-Ser linker is (Gly4Ser)6. In some embodiments, the Gly-Ser linker is (Gly4Ser)7. In some embodiments, the Gly-Ser linker is (Gly4Ser)8. In some embodiments, the Gly-Ser linker is (Gly4Ser)9. In some embodiments, the Gly-Ser linker is (Gly4Ser) 10 is.

[0113] In some embodiments, the second linker comprises a polypeptide linker. In some embodiments, the second linker comprises a plurality of glycine residues. In some embodiments, the second linker comprises a polypeptide linker having the amino acid sequence GGGGSGGGG. In some embodiments, the second linker comprises a polypeptide linker having the amino acid sequence GGGGEGGGG.

[0114] manifold In some embodiments, an immunomodulatory polypeptide or metal hydroxide-binding polypeptide utilized in accordance with the present disclosure is a variant of a related reference polypeptide (eg, a wild-type polypeptide or a functional portion thereof).

[0115] In some embodiments, the variant exhibits at least 70% identity to the reference polypeptide, hi some embodiments, the variant exhibits at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the reference polypeptide.

[0116] In some embodiments, a variant comprises one or more conservative or non-disruptive modifications (e.g., substitutions, deletions, or additions) compared to its reference. In some embodiments, a variant does not comprise a disruptive modification (e.g., substitution, deletion, or addition) such that the immunomodulatory polypeptide retains one or more functional properties of the reference. In some embodiments, retaining means that the immunomodulatory polypeptide exhibits equivalent activity (e.g., signaling ability or binding) compared to an appropriate reference standard (e.g., a wild-type immunomodulatory polypeptide). For example, in some such embodiments, the immunomodulatory polypeptide retains at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more activity compared to an appropriate reference standard (e.g., a wild-type immunomodulatory polypeptide).

[0117] metal hydroxide In some embodiments, the present disclosure provides a phosphorylated fusion polypeptide comprising an immunomodulatory polypeptide and a metal hydroxide-binding polypeptide, wherein the phosphorylated fusion polypeptide forms a complex with a metal hydroxide when exposed to the metal hydroxide. The complex is formed through adsorption of the phosphorylated fusion polypeptide to the metal hydroxide. While not wishing to be bound by any theory, it is hypothesized that adsorption of the phosphorylated fusion polypeptide to the metal hydroxide occurs by ligand exchange. Ligand exchange is, for example, the substitution or exchange of surface hydroxyls with another ligand. In some embodiments, the substitution or exchange of surface hydroxyl groups occurs with hydroxyl substituents (e.g., phosphate groups).

[0118] In some embodiments, a metal hydroxide is a substance containing at least one hydroxyl group bound to a metal. According to the present disclosure, in some embodiments, the metal hydroxide can adsorb a fusion polypeptide containing a hydroxyl group-substituted moiety. In some embodiments, the hydroxyl group-substituted moiety is a phosphate group.

[0119] In some embodiments, the metal hydroxide is selected based on its inherent qualities or properties. In some embodiments, the metal hydroxide is selected for its biocompatibility when used in a subject (e.g., a mammal, e.g., a human). In some embodiments, the metal hydroxide is aluminum hydroxide (e.g., alum). In some embodiments, the metal hydroxide is iron hydroxide. Those skilled in the art will recognize that any number of metal hydroxides may be successfully utilized in accordance with the present disclosure.

[0120] Phosphorylated fusion polypeptide preparations In particular, the present disclosure provides preparations of fusion polypeptides, and particularly phosphorylated fusion polypeptides, having particularly desired characteristics (e.g., the level of purity and / or degree of phosphorylation of such phosphorylated fusion polypeptides, e.g., the average degree of phosphorylation within an achieved preparation and / or the distribution of particular phosphoforms of the fusion polypeptide).

[0121] In particular, the present disclosure identifies sources of problems with certain methods for producing related fusion polypeptides, including, for example, their ability to produce preparations with a suitable degree and / or consistency (e.g., homogeneity) of phosphorylation and / or their ability to produce preparations of sufficient purity. The present disclosure provides particularly highly pure preparations of phosphorylated fusion polypeptides, including highly pure preparations characterized by a particular degree and / or consistency of phosphorylation.

[0122] Without being bound by any particular theory, the present disclosure notes that an excessively high or excessively low degree of phosphorylation can negatively affect the activity of a fusion polypeptide preparation.

[0123] The present disclosure provides particularly useful methods for producing phosphorylated forms of fusion polypeptides. In some embodiments, the present disclosure provides methods for producing phosphorylated forms of the fusion polypeptides disclosed herein by contacting the fusion polypeptide with a kinase. Thus, the production methods described herein provide predetermined fusion polypeptide preparations characterized by a particularly desired degree of phosphorylation. Alternatively or additionally, in some embodiments, the provided techniques achieve particularly high yield production of the desired preparations described herein (e.g., having the level and / or consistency and / or degree of purity of phosphorylation described herein). In some embodiments, the present invention provides methods for producing highly pure preparations of phosphorylated forms of fusion polypeptides.

[0124] In some embodiments, a highly pure phosphorylated fusion polypeptide preparation comprises more phosphorylated fusion polypeptide than non-phosphorylated fusion polypeptide. In some embodiments, a highly pure phosphorylated fusion polypeptide preparation comprises primarily the phosphorylated fusion polypeptide being compared. In some embodiments, a highly pure phosphorylated fusion polypeptide preparation comprises at least 90% phosphorylated fusion polypeptide, e.g., at least 91%, e.g., at least 92%, e.g., at least 93%, e.g., at least 94%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99% phosphorylated fusion polypeptide.

[0125] In some embodiments, the fusion polypeptide preparation comprises an unphosphorylated form of the fusion polypeptide. In some embodiments, the fusion polypeptide preparation comprises a phosphorylated form of the fusion polypeptide. In some embodiments, the fusion polypeptide preparation comprises a mixture of both the unphosphorylated and phosphorylated forms of the fusion polypeptide.

[0126] In some embodiments, a fusion polypeptide preparation is a preparation comprising a fusion polypeptide in which one or more characterization attributes have been evaluated and determined to meet release and / or acceptance criteria (e.g., as described herein). Examples of such product quality attributes include, but are not limited to, degree of phosphorylation and / or phosphorylation heterogeneity.

[0127] In some embodiments, fusion polypeptides having phosphorylated and unphosphorylated forms described herein are produced in host cells using techniques for exogenous expression.

[0128] In some embodiments, the fusion polypeptide preparation comprises a phosphorylated fusion polypeptide. In some embodiments, the fusion polypeptide preparation is a highly purified preparation of a phosphorylated form of a fusion polypeptide. In some embodiments, the fusion polypeptide preparation comprises a mixture of both unphosphorylated and phosphorylated forms of a fusion polypeptide. In some embodiments, the fusion polypeptide preparation comprises more phosphorylated than unphosphorylated fusion polypeptides. In some embodiments, the phosphorylated fusion polypeptide preparation comprises fusion polypeptides with various degrees of phosphorylation as described hereinabove. In some embodiments, the fusion polypeptide preparation is prepared in a Tris buffer at about pH 7 to about pH 8. In some embodiments, the fusion polypeptide preparation comprises a salt (e.g., NaCl).

[0129] Production of phosphorylated fusion polypeptides Expression in host cells In some embodiments of the present disclosure, the fusion polypeptide is produced by production in a host cell, e.g., a mammalian cell. Typically, such a host cell (e.g., such a mammalian cell) will be engineered to express the fusion polypeptide. Those of skill in the art will be familiar with various techniques for introducing exogenous gene sequences (e.g., encoding a fusion polypeptide and / or a kinase) into a host cell, e.g., a mammalian host cell, for expression therethrough.

[0130] For example, in some embodiments, a polynucleotide (e.g., DNA or RNA) encoding a fusion polypeptide of the present disclosure can be prepared, e.g., for introduction into a host cell. For example, the sequence encoding the fusion polypeptide can be excised from DNA using a restriction enzyme, amplified from a plasmid or genomic polynucleotide sequence using, e.g., the polymerase chain reaction, or synthesized using chemical synthesis techniques. In some embodiments, a combination of known methods is utilized to prepare a recombinant polynucleotide encoding a fusion polypeptide of the present disclosure.

[0131] A recombinant polynucleotide encoding a fusion polypeptide of the present disclosure can be cloned into a vector capable of expressing the fusion polypeptide. Cloning can be performed by a variety of available methods (e.g., Gibson assembly, restriction enzyme digestion, and ligation, etc.). In some embodiments, the vector is a viral vector. In some embodiments, the vector is a non-viral vector. In some embodiments, the vector is a plasmid. In some embodiments, the vector is a transposon.

[0132] In some embodiments, an expression-capable vector comprises a recombinant polynucleotide encoding a fusion polypeptide of the present disclosure operably linked to a sequence(s) controlling expression of the polynucleotide (e.g., promoter, start signal, stop signal, polyadenylation signal, activator, repressor, etc.). In some embodiments, the regulatory sequence(s) controlling expression are selected to achieve a desired level of expression. In some embodiments, two or more expression-controlling sequences (e.g., promoters) are utilized. In some embodiments, two or more expression-controlling sequences (e.g., promoters) are utilized to achieve a desired level of expression of multiple recombinant polynucleotides encoding multiple polypeptides. In some embodiments, multiple recombinant polypeptides are expressed from the same vector (e.g., bicistronic, tricistronic, multicistronic). In some embodiments, multiple recombinant polypeptides are expressed, each expressed from a separate vector.

[0133] In some embodiments, an expression-enabling vector comprising a recombinant polynucleotide encoding a fusion polypeptide of the present disclosure is used to express the fusion polypeptide in a host cell.

[0134] Host cells can be selected from a variety of available and known host cells suitable for expression of the fusion polypeptides disclosed herein (e.g., human embryonic kidney (HEK) cells, suspension HEK293 cells, Chinese hamster ovary cells).

[0135] Various methods are available for introducing vectors into host cells. In some embodiments, vectors can be introduced into host cells using transfection. In some embodiments, transfection is accomplished using, for example, calcium phosphate transfection, lipofection, or polyethyleneimine-mediated transfection. In some embodiments, vectors can be introduced into host cells using transduction.

[0136] In some embodiments, a host cell (eg, a producer cell) is used to produce a phosphorylated form of a fusion polypeptide.

[0137] In some embodiments, host cells expressing the fusion polypeptide and / or kinase are cultured in single-use bioreactors (e.g., 50 L to 4000 L) or stainless steel bioreactors (e.g., 50 L to 4000 L). In some embodiments, the host cells are cultured at a temperature ranging from 30°C to 40°C. In some embodiments, the temperature is lower (e.g., 33°C) during the production phase. In some embodiments, the cell extract is harvested through a two- or three-stage filter, followed by a final sterile 0.22 μm filtration.

[0138] In some embodiments, a nucleic acid encoding a fusion polypeptide is introduced into a host cell such that the fusion polypeptide is expressed by the host cell. Alternatively or additionally, in some embodiments, a nucleic acid encoding a kinase that phosphorylates the fusion polypeptide is introduced into the host cell such that the host cell expresses the kinase. In many embodiments, as described herein, both a nucleic acid encoding a fusion polypeptide and a nucleic acid encoding a kinase that phosphorylates it are introduced into the same host cell; in some such embodiments, a single nucleic acid molecule may encode both.

[0139] In some embodiments, the nucleic acid molecule introduced into the cell is RNA (e.g., mRNA); in some such embodiments, the encoded polypeptide(s) (e.g., fusion polypeptide and / or kinase) are expressed from such RNA. Alternatively, or additionally, in some embodiments, the nucleic acid molecule introduced into the cell is DNA (e.g., single-stranded DNA or double-stranded DNA). In some embodiments, the nucleic acid is introduced into the cell such that the coding sequence is integrated into the host cell (e.g., into its genome); in some such embodiments, the encoded polypeptide(s) (e.g., fusion polypeptide and / or kinase) are expressed therefrom.

[0140] In some embodiments, nucleic acid molecules (e.g., nucleic acid molecules encoding fusion polypeptides and / or kinases) introduced into cells include one or more expression elements that can, for example, control the expression of such encoded polypeptide(s). Alternatively, or additionally, in some embodiments, nucleic acid molecules (e.g., nucleic acid molecules encoding fusion polypeptides and / or kinases) introduced into cells can be designed or intended to be associated (e.g., by integration) with one or more regulatory elements in the host cell.

[0141] In some embodiments, vectors (e.g., transposons) containing sequences encoding the fusion polypeptides and / or kinases described herein are used to express the fusion polypeptides and / or kinases in host cells.

[0142] In some embodiments, host cells can be selected from a variety of available and known host cells suitable for expression of the fusion polypeptides disclosed herein (e.g., human embryonic kidney (HEK) cells, suspension HEK293 cells, Chinese hamster ovary cells). In some embodiments, the host cells are mammalian cells.

[0143] Various methods are available for introducing nucleic acids (e.g., vectors, e.g., expression vectors) into host cells. In some embodiments, nucleic acids may be introduced into host cells using transfection. In some embodiments, transfection is completed using, for example, calcium phosphate transfection, lipofection, or polyethyleneimine-mediated transfection. In some embodiments, nucleic acids may be introduced into host cells using transduction. In some embodiments, nucleic acids may be introduced into host cells using electroporation. In some embodiments, nucleic acids may be introduced into host cells using particle delivery, e.g., polymer particle delivery, lipid particle delivery, gold particle delivery, etc.

[0144] phosphorylation In some embodiments, the present disclosure provides methods for producing a phosphorylated form of a fusion polypeptide disclosed herein by contacting the fusion polypeptide with a kinase. In some embodiments, a nucleic acid, e.g., a nucleic acid encoding the fusion polypeptide and / or the kinase, is introduced into a host cell. In some embodiments, the fusion polypeptide is contacted with the kinase by coexpression of the fusion polypeptide and the kinase in the host cell. In some embodiments, coexpression is achieved by introducing into the host cell two vectors: one vector containing a recombinant polynucleotide encoding the fusion polypeptide and another vector containing a recombinant polynucleotide encoding the kinase. In some embodiments, coexpression is achieved by introducing a single multicistronic (e.g., bicistronic) vector containing multiple recombinant polynucleotides (e.g., transposons). In some embodiments, a recombinant polynucleotide encodes the fusion polypeptide and a recombinant polynucleotide encodes the kinase. In some embodiments, the transformed host cell is cultured after introduction of the vector (e.g., transposon) into the host cell. Without wishing to be bound by any theory, coexpression of the fusion polypeptide and the kinase in the host cell may allow the kinase to contact and phosphorylate the fusion polypeptide.

[0145] In some embodiments, coexpression is achieved by introducing two vectors into a host cell: one vector containing a recombinant polynucleotide encoding a fusion polypeptide and another vector containing a recombinant polynucleotide encoding a kinase. In some embodiments, the two vectors are introduced such that the ratio of vector encoding the fusion polypeptide to vector encoding the kinase introduced into the host cell is optimized to achieve a desired relative level of expression of the fusion polypeptide relative to the kinase. In some embodiments, the ratio of vector encoding the fusion polypeptide to vector encoding the kinase is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, or 100:1.

[0146] In some embodiments, co-expression is achieved by introducing into the host cell a single vector containing both a recombinant polynucleotide encoding the fusion polypeptide and a recombinant polypeptide encoding the kinase (a bicistronic vector). In some embodiments, co-expression is achieved by introducing into the host cell a single transposon containing both a recombinant polynucleotide encoding the fusion polypeptide and a recombinant polypeptide encoding the kinase. In some embodiments, the transposon is a DNA transposon. In some embodiments, the transposon, or a portion thereof (e.g., including the nucleotides encoding the fusion polypeptide and the nucleotides encoding the kinase), is integrated into the host cell genome by an integrase (i.e., by an integrase enzyme, e.g., a DDE / D integrase enzyme). In some embodiments, the integrase is delivered to the host cell as mRNA. In some embodiments, the integrase is a PiggyBac enzyme. In some embodiments, the integrase is a Leap-In transposase. In some embodiments, the transposon, or a portion thereof, is not integrated into the genome by random integration. In some embodiments, a single copy of a polynucleotide encoding a fusion polypeptide of the present disclosure is integrated into specific multiple host cell genomic loci. In some embodiments, integration of a polynucleotide encoding a fusion polypeptide of the present disclosure is irreversible. Irreversible integration of the fusion polypeptide into the host cell genome can ensure stable integration, allowing for the generation of highly stable cell lines. In some embodiments, the recombinant polynucleotide encoding the fusion polypeptide and the recombinant polynucleotide encoding the kinase are operably linked to sequence(s) that control expression (e.g., promoter, start signal, stop signal, polyadenylation signal, activator, repressor, etc.). In some embodiments, the sequence(s) that control expression are selected to achieve a desired level of expression.In some embodiments, multiple regulatory nucleotide sequences (e.g., promoters) are utilized to control expression to achieve a desired expression ratio of fusion polypeptide to kinase. In some embodiments, the ratio is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, or 100:1. In some embodiments, the regulatory nucleotide sequence is a promoter. In some embodiments, a particular ratio (e.g., 2:1, 4:1, 8:1, or 15:1) is achieved using a single transposon with two promoters to express the fusion polypeptide and kinase. In some embodiments, a single independent transposon contains promoters of different strengths to generate a desired ratio (e.g., 8:1). In some embodiments, the promoter is a CMV or EF1a promoter. In some embodiments, the fusion polypeptide is under the control of a CMV promoter or an EF1a promoter. In some embodiments, the promoter is an SV40 or Ubc promoter. In some embodiments, the kinase is under the control of an SV40 promoter or a Ubc promoter. In some embodiments, the ratio of fusion polypeptide to kinase is 8:1.

[0147] In some embodiments, transformed host cells (i.e., host cells into which a nucleic acid, such as a nucleic acid encoding a fusion polypeptide and / or a kinase, has been introduced) are cultured after such transformation, e.g., to allow for expression of the recombinant polynucleotide. In some embodiments, transformed host cells are cultured for at least 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72 hours, or more. In some embodiments, transformed host cells are cultured for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more days. Transformed host cells are cultured under growth conditions (e.g., temperature, carbon dioxide level, growth medium) according to the requirements of the selected host cells. One of skill in the art will recognize that culture conditions for selected host cells are well known in the art. In some embodiments, the host cells secrete the phosphorylated form of the fusion polypeptide into the cell extract. In some embodiments, the host cells may exhibit increased secretion of the phosphorylated fusion polypeptide into the cell extract compared to the non-phosphorylated fusion polypeptide. In some embodiments, the host cells secrete comparable levels of the phosphorylated and non-phosphorylated fusion polypeptide into the cell extract. In some embodiments, the cell extract comprises the non-phosphorylated form of the fusion polypeptide. In some embodiments, the cell extract comprises the phosphorylated form of the fusion polypeptide. In some embodiments, the cell extract comprises a mixture of both the non-phosphorylated and phosphorylated forms of the fusion polypeptide. In some embodiments, the cell extract comprises more of the phosphorylated form of the fusion polypeptide than the non-phosphorylated form of the fusion polypeptide.

[0148] In some embodiments, the host cells secrete the phosphorylated form of the fusion polypeptide into cell extracts, but do not secrete the kinase, hi some embodiments, the host cells do not secrete the kinase, or secrete only small amounts of the kinase.

[0149] In some embodiments, the host cell extract comprises a phosphorylated form of the fusion polypeptide. In some embodiments, the host cell extract comprises host cell proteins and / or host cell nucleotides.

[0150] In some embodiments, the phosphorylated form of the fusion polypeptide is harvested from the transformed host cells and clarified by centrifugation.

[0151] In some embodiments the transformed host cell is characterized in that a culture thereof produces the fusion protein at a titer of at least 200 mg / L, such as at least 250 mg / L, for example, at least 300 mg / L, such as at least 350 mg / L, for example, at least 400 mg / L, such as at least 450 mg / L, for example, at least 500 mg / L, such as at least 550 mg / L, for example, at least 600 mg / L, such as at least 650 mg / L, for example, at least 700 mg / L, such as at least 750 mg / L, for example, at least 800 mg / L, such as at least 850 mg / L, for example, at least 900 mg / L, such as at least 950 mg / L, for example, at least 1 g / L or more.

[0152] In some embodiments, one or more serine residues at position 43, 281, 306, 311, 316, 365, or 481 of SEQ ID NO:2 are phosphorylated. In some embodiments, one or more serine residues at position 43, 154, 281, 306, 311, 316, 365, or 481 of SEQ ID NO:2 are phosphorylated. In some embodiments, one or more serine residues at position 43, 154, 168, 281, 306, 311, 316, 365, or 481 of SEQ ID NO:2 are phosphorylated. In some embodiments, one or more serine residues at position 43, 154, 168, 281, 306, 311, 316, 365, 406, or 481 of SEQ ID NO:2 are phosphorylated. In some embodiments, at least the serine residue at position 481 of SEQ ID NO:2 is phosphorylated.

[0153] purification In some embodiments, the disclosure provides techniques (e.g., manufacturing techniques) that are or include purification methods, e.g., methods that include one or more purification steps. In some embodiments, the phosphorylated form of the fusion protein is purified from an extract of a cell described herein.

[0154] In some embodiments, the purification step can include the removal of commonly abnormal products from the cell extract, such as residual proteins, host cell contaminants (e.g., host DNA and / or proteins, etc.).

[0155] In some embodiments, the production method, including one or more purification steps, results in a highly pure preparation of the phosphorylated form of the fusion polypeptide (see, e.g., Examples 10-11). In some embodiments, the highly pure preparation of the phosphorylated form of the fusion polypeptide contains reduced host cell protein equivalent to the cell extract described above. In some embodiments, such a highly pure preparation is free of any host cell protein. In some embodiments, the highly pure preparation contains less than 100 ng / mg of host cell protein, e.g., less than 50 ng / mg, e.g., less than 40 ng / mg, e.g., less than 30 ng / mg, e.g., less than 20 ng / mg, e.g., less than 10 ng / mg, e.g., less than 9 ng / mg, e.g., less than 8.5 ng / mg of host cell protein. In some embodiments, the highly pure preparation of the phosphorylated form of the fusion polypeptide contains reduced host cell DNA equivalent to the cell extract described above. In some embodiments, such a highly pure preparation is free of any host cell DNA. In some embodiments, a highly pure preparation comprises less than 10 pg / mg of host cell DNA, such as less than 9 pg / mg, for example, less than 8 pg / mg, such as less than 7 pg / mg, for example, less than 6 pg / mg, such as less than 5 pg / mg, for example, less than 4 pg / mg, such as less than 3 pg / mg, for example, less than 2 pg / mg, such as less than 1.5 pg / mg, for example, less than 1 pg / mg, such as less than 0.9 pg / mg, for example, less than 0.8 pg / mg, for example, less than 0.7 pg / mg of host cell DNA.

[0156] In some embodiments, the high purity preparation contains low levels of in-process compounds. In some embodiments, the in-process compounds can be tropolone, Pluronic, PDMS, octamethylcyclotetrasiloxane D4, TDAO, and / or Fam20C). In some embodiments, the high purity preparation contains less than 1 mg / mL TDAO, e.g., less than 0.9 mg / mL TDAO, e.g., less than 0.8 mg / mL TDAO, e.g., less than 0.7 mg / mL TDAO, e.g., less than 0.6 mg / mL TDAO, e.g., less than 0.5 mg / mL TDAO, e.g., less than 0.4 mg / mL TDAO, e.g., less than 0.3 mg / mL TDAO. In some embodiments, a highly pure preparation has less than 5000 ng of Fam20C / mg (IL-12 fusion polypeptide), such as less than 4000 ng of Fam20C / mg (IL-12 fusion polypeptide), for example, less than 3000 ng of Fam20C / mg (IL-12 fusion polypeptide), for example, less than 2500 ng of Fam20C / mg (IL-12 fusion polypeptide), for example, less than 2000 ng of Fam20C / mg (IL-12 fusion polypeptide), for example, less than 1800 ng of Fam20C / mg (IL-12 fusion polypeptide), for example, less than 1000 ng of Fam20C / mg (IL-12 fusion polypeptide), for example, less than 75 ... 100 ng Fam20C / mg (IL-12 fusion polypeptide), for example, less than 500 ng Fam20C / mg (IL-12 fusion polypeptide), for example, less than 300 ng Fam20C / mg (IL-12 fusion polypeptide), for example, less than 200 ng Fam20C / mg (IL-12 fusion polypeptide), for example, less than 100 ng Fam20C / mg (IL-12 fusion polypeptide), for example, less than 80 ng Fam20C / mg (IL-12 fusion polypeptide), for example, less than 70 ng Fam20C / mg (IL-12 fusion polypeptide), for example, less than 60 ng Fam20C / mg (IL-12 fusion polypeptide).

[0157] In some embodiments, the phosphorylated form of the fusion polypeptide can be purified by including one or more chromatographic purification steps. In some embodiments, one or more conventional chromatographic steps are used. In some embodiments, the conventional chromatographic steps utilize anion or cation exchange, hydrophobic interaction, or hydroxyapatite chromatography.

[0158] In some embodiments, the phosphorylated form of the fusion polypeptide can be purified by including one or more purification steps selected from an ionic chromatography step (e.g., an anionic chromatography step) and a hydrophobic interaction chromatography step, thus resulting in the separation of the phosphorylated fusion polypeptide from impurities.

[0159] Those skilled in the art will be familiar with various purification (e.g., chromatography) matrices and formats thereof that may be utilized in accordance with the present disclosure. For example, in some embodiments, beads, particles, microspheres, resins, etc. may be utilized. In some embodiments, the matrix utilized for purification (e.g., for chromatography) has properties that allow for a different retention time for the fusion polypeptide relative to any other undesired components in the fusion polypeptide preparation in accordance with the present disclosure.

[0160] In some embodiments, the phosphorylated form of the fusion polypeptide is not purified by an affinity-based purification method, e.g., in some embodiments, the purification techniques provided do not utilize affinity chromatography.

[0161] In some embodiments, the phosphorylated form of the fusion polypeptide can be eluted from the solid substrate. In some embodiments, elution can be performed using specific elution. For example, in some embodiments, specific elution is accomplished by triggering the polypeptide-substrate complex with an agent(s) that would compete for complexation with either the substrate or the polypeptide, releasing the polypeptide into solution. In some embodiments, elution can be performed using non-specific elution. For example, in some embodiments, general elution is accomplished by manipulating solvent or buffer conditions (e.g., increasing the concentration of a buffer, such as an imidazole buffer) to decrease the association rate constant, resulting in dissociation of the polypeptide from the substrate.

[0162] First Chromatography Step In some embodiments, methods according to the present disclosure include at least one ion chromatography step (e.g., an anion chromatography step). In some embodiments, methods according to the present disclosure include at least one anion chromatography step.

[0163] In some embodiments, the first chromatography step is a capture step (e.g., an anion chromatography capture step). Without wishing to be bound by any theory, phosphorylation of a polypeptide confers variability in the charge of the polypeptide, thereby enabling the separation of differentially phosphorylated polypeptides using ion exchange chromatography (e.g., anion exchange chromatography). Anion exchange chromatography is a form of ion exchange in which negatively charged biomolecules (e.g., phosphorylated forms of the fusion polypeptides disclosed herein) bind to a positively charged solid substrate (e.g., a resin). The positively charged solid substrate thereby captures the negatively charged fusion polypeptide from the cell extract while simultaneously removing positively charged impurities and fusion polypeptide aggregates (because the aggregates are likely to have a less negative charge) from the cell extract.

[0164] In some embodiments, anion exchange chromatography can be used to separate polypeptides having different numbers of phosphorylated amino acid residues (e.g., differentially phosphorylated polypeptides). Anion exchange chromatography can enrich a fusion polypeptide preparation for highly phosphorylated species (e.g., fusion polypeptides having more than six phosphorylation sites). Thus, in some embodiments, a preparation having a high concentration of phosphorylated fusion polypeptide and a low concentration of positively charged impurities is generated through the use of an anion exchange chromatography step (e.g., as a first step).

[0165] In some embodiments, purifying the phosphorylated form of the fusion polypeptide from the cell extract comprises an anion chromatography capture step. In some embodiments, the anion chromatography capture step is the first capture step.

[0166] In some embodiments, anion exchange chromatography utilizes an anion exchange resin bearing covalently bound positively charged groups, e.g., quaternary amino groups. Commercially available anion exchange resins include Q Sepharose, DEAE Sepharose, TMAE, GigaCap Q 650M, and 650S. Binding of a negatively charged biomolecule (e.g., a phosphorylated form of a fusion polypeptide) to an anion exchange material is, in some embodiments, accomplished by exposing the negatively charged biomolecule to the resin under appropriate conditions (e.g., pH / conductivity), thereby immobilizing the biomolecule on the anion exchange resin via ionic interactions between the negatively charged biomolecule and the charged group(s) of the ion exchange material.

[0167] The wash step may involve passing an appropriate buffer through the chromatography resin to wash away unwanted materials, such as host cell proteins or host cell nucleotides. In some embodiments, the wash buffer may contain various conditions, e.g., pH, conductivity, aimed at dissociating impurities nonspecifically bound to the chromatography resin. In some embodiments, the wash step utilizes a mixture of equilibration and elution buffers.

[0168] The phosphorylated form of the fusion polypeptide can be eluted from the solid substrate (e.g., a positively charged resin) using elution. In some embodiments, the negatively charged agent (e.g., the phosphorylated form of the fusion polypeptide) is eluted using a buffer that reduces the interaction between the anion exchange resin and the negatively charged agent (e.g., the phosphorylated fusion polypeptide). In some embodiments, such an elution buffer can have a higher concentration of salt and / or a different pH to facilitate dissociation of the negatively charged agent from the chromatography resin.

[0169] In some embodiments, a gradient elution buffer (e.g., a buffer with increasing salt concentration) is used to elute from an ion exchange (e.g., anion exchange) column. In some such embodiments, the use of such a gradient can allow for the separation of differentially phosphorylated polypeptides (i.e., achieve the separation of different phosphoforms).

[0170] In some embodiments, the buffer is, for example, a Tris buffer. In some embodiments, a linear gradient of Tris buffer is utilized. In some embodiments, the linear gradient of Tris buffer comprises a linear gradient from 20 mM Tris, pH 7.1 to 20 mM Tris, 1 M NaCl, pH 7.1 over a predefined period of time. In some embodiments, the linear gradient is performed over a period of 1 minute, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 22 minutes, 24 minutes, 26 minutes, 28 minutes, 30 minutes, 32 minutes, 34 minutes, 36 minutes, 38 minutes, 40 minutes, or more. In some embodiments, the first anion chromatography capture step utilizes a Tris buffer. In some embodiments, the first anion chromatography capture step is performed at a pH of about 6 to about 9, e.g., about 7 to about 8.

[0171] In some embodiments, the first anion chromatography capture step is carried out using capture beads. In some embodiments, the first step capture beads have a diameter of at least 50 μm, for example, at least 55 μm, for example, at least 60 μm, for example, at least 65 μm, for example, at least 70 μm, for example, at least 75 μm. In some embodiments, the first capture beads are GigaCap Q 650M.

[0172] In some embodiments, the first anion chromatography capture step includes an immobilization step (e.g., binding the phosphorylated fusion polypeptide to the chromatography column), a pre-elution wash step, and an elution step (e.g., eluting the phosphorylated fusion polypeptide). In some embodiments, resin beads having an average particle size of about 50 to about 100 micrometers, e.g., 75 micrometers, are utilized to immobilize the phosphorylated fusion polypeptide. In some embodiments, an immobilization composition having a low salt concentration is utilized during immobilization of the phosphorylated fusion polypeptide to the chromatography column (e.g., 0 mM sodium chloride is utilized). In some embodiments, a pre-elution composition having an intermediate salt concentration, comparable to the salt concentrations of the immobilization and elution compositions (e.g., 215 mM sodium chloride is utilized), is utilized during the pre-elution wash step. In some embodiments, an elution composition having a high salt concentration is utilized during elution of the phosphorylated fusion polypeptide from the chromatography column (e.g., 350 mM sodium chloride is utilized). In some embodiments, the first anion chromatography capture step is carried out at a pH in the range of about 7 to about 8 (e.g., 7.4). In some embodiments, a flow rate of 200 to 400 cm / h is used (e.g., 300 cm / h).

[0173] Second Chromatography Step In some embodiments, the method according to the present disclosure includes at least one hydrophobic interaction chromatography step. In some embodiments, the hydrophobic interaction step is performed after the first anion chromatography step. Without being bound by theory, the phosphorylated form of the fusion protein according to the present disclosure has low hydrophobicity (e.g., due to its degree of phosphorylation and therefore its charge) that can be used to separate it from hydrophobic impurities. In some embodiments, hydrophobic host cell impurities (e.g., host cell proteins) and / or fusion polypeptide aggregates are separated from a preparation of the phosphorylated form of the fusion polypeptide. In some embodiments, the preparation (e.g., a high-purity preparation) contains less than 5% aggregated fusion polypeptide, less than 4%, less than 3%, less than 3%, less than 2%, less than 1%, e.g., less than 0.9%, e.g., less than 0.8% aggregated fusion polypeptide.

[0174] A hydrophobic interaction step may, in some embodiments, separate product or process-related impurities, such as host cell proteins or aggregation products (e.g., fusion polypeptide aggregates), from the phosphorylated form of the fusion polypeptide based on differential hydrophobic interactions between the phosphorylated fusion polypeptide and the impurities and hydrophobic substances. Such a step may, in some embodiments, be referred to as a polishing step.

[0175] Examples of hydrophobic interaction resins include, but are not limited to, hydrophobic ligands, such as alkyl groups ranging from 2 to 8 carbon atoms, or aryl groups, such as phenyl. Binding of a negatively charged agent (e.g., a phosphorylated form of a fusion polypeptide) to a hydrophobic interaction resin, in some embodiments, involves exposing the biomolecule to the resin under appropriate conditions (pH / conductivity), thereby immobilizing the biomolecule to the hydrophobic resin via hydrophobic interactions between the biomolecule and the non-polar groups of the hydrophobic interaction material. Hydrophobic interaction binding typically occurs at high salt concentrations (e.g., 1 to 1.8 M ammonium sulfate). In some embodiments, the phosphorylated form of the fusion polypeptide is immobilized at high salt concentrations (e.g., 1.4 M sodium sulfate). In some embodiments, the phosphorylated form of the fusion polypeptide is eluted with a linear gradient ranging from 1.4 M ammonium sulfate to 0 M ammonium sulfate.

[0176] The wash step may involve passing a suitable buffer through the chromatography resin to wash away unwanted materials, such as less hydrophobic host cell proteins. In some embodiments, the wash buffers may have different pHs to facilitate dissociation of less hydrophobic agents from impurities nonspecifically bound to the chromatography resin. In some embodiments, the wash step utilizes a mixture of equilibration and elution buffers.

[0177] The phosphorylated form of the fusion polypeptide can be eluted from the solid substrate (e.g., a hydrophobic resin) using elution. In some embodiments, the less hydrophobic agent (e.g., the phosphorylated form of the fusion polypeptide) is eluted from the hydrophobic resin using a buffer that reduces the interaction between the hydrophobic interaction resin and the negatively charged agent (e.g., the phosphorylated fusion polypeptide). In some embodiments, such an elution buffer can have a lower concentration of salt or a change in pH that promotes dissociation of the biomolecule from the chromatography resin. In some embodiments, the phosphorylated form of the fusion polypeptide is eluted with a low salt concentration (e.g., 750 mM sodium sulfate).

[0178] In some embodiments, the hydrophobic interaction chromatography step includes an immobilization step (e.g., binding the phosphorylated fusion polypeptide to a chromatography column) and an elution step (e.g., eluting the phosphorylated fusion polypeptide). In some embodiments, resin beads having an average particle size of about 50 to about 100 micrometers, e.g., 75 micrometers, are utilized to immobilize the phosphorylated fusion polypeptide. In some embodiments, an immobilization composition having a high salt concentration is used during immobilization of the phosphorylated fusion polypeptide to the chromatography column (e.g., 1.4 M ammonium sulfate is used). In some embodiments, an elution composition having a low salt concentration is used during elution of the phosphorylated fusion polypeptide from the chromatography column (e.g., 740 mM ammonium sulfate is used). In some embodiments, the hydrophobic interaction chromatography step is performed at a pH in the range of about 7 to about 8 (e.g., 7.4). In some embodiments, a flow rate of 200 to 350 cm / h is used (e.g., 275 cm / h).

[0179] Third Chromatography Step In some embodiments, methods according to the present disclosure include a first anion chromatography step and a second anion chromatography step. In some embodiments, methods according to the present disclosure include a first anion chromatography step and a second anion chromatography step, wherein the first anion chromatography step is a capture step and the second anion chromatography step is a polishing step. In some embodiments, the second anion chromatography step is performed after the hydrophobic interaction chromatography step.

[0180] In some embodiments, the method according to the present disclosure comprises the steps of: i) a first anion chromatography step; ii) a hydrophobic interaction chromatography step; and iii) Second Anion Chromatography Step Includes:

[0181] In some embodiments, the second anion chromatography step is performed using capture beads. In some embodiments, the capture beads of the second step have a diameter of at most 50 μm, at most 45 μm, at most 40 μm, or at most 35 μm. In some embodiments, the first capture beads are GigaCap Q 650S.

[0182] In some embodiments, the second anion chromatography capture step includes an immobilization step (e.g., binding the phosphorylated fusion polypeptide to the chromatography column), a pre-elution wash step, and an elution step (e.g., eluting the phosphorylated fusion polypeptide). In some embodiments, resin beads having an average particle size of about 10 to about 50 micrometers, e.g., 35 micrometers, are utilized to immobilize the phosphorylated fusion polypeptide. In some embodiments, an immobilization composition having a low salt concentration is utilized during immobilization of the phosphorylated fusion polypeptide to the chromatography column (e.g., 0 mM sodium chloride is utilized). In some embodiments, a pre-elution composition having an intermediate salt concentration, comparable to the salt concentrations of the immobilization and elution compositions (e.g., 274 mM sodium chloride is utilized), is utilized during the pre-elution wash step. In some embodiments, an elution composition having a high salt concentration is utilized during elution of the phosphorylated fusion polypeptide from the chromatography column (e.g., 355 mM sodium chloride is utilized). In some embodiments, the first anion chromatography capture step is carried out at a pH in the range of about 7 to about 8 (e.g., 7.3). In some embodiments, a flow rate of 200 to 400 cm / h is used (e.g., 300 cm / h).

[0183] Optional additional step(s) In some embodiments, the method according to the present disclosure includes a virus inactivation or virus removal step. In some embodiments, the virus inactivation or virus removal step is performed before or after any of the chromatography steps described herein above. In one embodiment, the virus inactivation or virus removal step is performed before the first chromatography step (e.g., the first anion chromatography step). In some embodiments, the virus inactivation step includes pH inactivation or chemical inactivation (e.g., via the use of a chemical agent, e.g., a surfactant). In some embodiments, the virus inactivation step includes utilizing a detergent because IL12-ABP is sensitive to low pH and may aggregate, without being bound by theory. In some embodiments, the virus inactivation step includes utilizing a detergent selected from myristyldimethylamine N-oxide, TDAO, Triton X-100, or polysorbate. In some embodiments, the virus removal step includes a filtration step.

[0184] Characterization In particular, in some embodiments, the present disclosure provides preparations of fusion polypeptides (e.g., phosphorylated or non-phosphorylated) or complexes thereof and / or comprising such fusion polypeptides and metal hydroxides, or techniques for characterizing preparations of such complexes.

[0185] In some embodiments, one or more characterizations may be performed during and / or after one or more steps of the manufacturing processes (e.g., purification processes) described herein. In some embodiments, a particular preparation process may be modified or terminated in light of a characterization (e.g., if a particular preparation does not meet one or more specifications). In some embodiments, a characterization may involve assessment of one or more of metal hydroxide retention, degree of phosphorylation, phosphorylation heterogeneity, signaling activity, and / or efficacy. See exemplary suitable characterization assays under "Data Analysis" in Example 3.

[0186] In some embodiments, after purification of the phosphorylated fusion polypeptides (e.g., by one or more chromatography steps), the amount of each phosphorylated fusion polypeptide is measured. In some embodiments, the amount of each phosphorylated fusion polypeptide is measured according to one or more of a variety of methods available in the art. In some embodiments, for example and without limitation, the phosphorylated fusion polypeptide is measured using a malachite green assay, analytical ion exchange, spectrophotometer, colorimetric assay, intact mass analysis by mass spectrometry, and / or Western blot. See exemplary suitable characterization assays under "Data Analysis" in Example 3.

[0187] Exemplary characterization of phosphate content In some embodiments, the degree of phosphorylation (e.g., of a fusion polypeptide of the present disclosure) is characterized. Various methods for measuring the degree of phosphorylation (e.g., the average number of phosphate molecules per polypeptide) are available. For example, in some embodiments, the degree of phosphorylation can be determined by a colorimetric method. In some embodiments, the colorimetric method is or includes a malachite green assay. Without wishing to be bound by any theory, the malachite green assay is based on the quantification of the green complex formed between malachite green molybdate and free orthophosphate, which can be measured (using a spectrophotometer or plate reader).

[0188] In some embodiments, the degree of phosphorylation (e.g., the average number of phosphate molecules per polypeptide) is about 4-12, about 5-11, about 6-10, about 7-9, or about 7.5-8.5 (see, e.g., exemplary degrees of phosphorylation in Example 10). In some embodiments, the degree of phosphorylation (e.g., average number of phosphate molecules per polypeptide) is 5.0, 5.5, 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, 7.10, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.5, or 11. In some embodiments, the degree of phosphorylation (e.g., the average number of phosphate molecules per polypeptide) is 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 7.10, 8.0, or 8.1.

[0189] In some embodiments, the phosphorylation heterogeneity of fusion polypeptides of the present disclosure and / or preparations thereof is characterized. In some embodiments, phosphorylation heterogeneity is a measure of the degree of phosphorylation within a given preparation of the fusion polypeptide. In some embodiments, phosphorylation heterogeneity is a measure of the degree of phosphorylation across multiple preparations of the fusion polypeptide. In some embodiments, phosphorylation heterogeneity is a measure of the location of a particular phosphate group on the polypeptide within a given preparation of the fusion polypeptide. In some embodiments, phosphorylation heterogeneity is a measure of the location of a particular phosphate group on the polypeptide across multiple preparations of the fusion polypeptide.

[0190] A variety of techniques are available for measuring phosphorylation heterogeneity, for example, in some embodiments, the degree of phosphorylation can be determined by chromatographic methods, as described herein above.

[0191] In some embodiments, the differentially phosphorylated polypeptide is dephosphorylated. In some embodiments, dephosphorylation involves the use of a phosphatase (e.g., lambda phosphatase). In some embodiments, the fusion polypeptide is incubated with the phosphatase for a period of time and at a temperature that allows for the activity of the phosphatase and dephosphorylation of the fusion polypeptide. In some embodiments, dephosphorylation occurs at an incubation temperature of approximately 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, or higher. In some embodiments, dephosphorylation is performed for an incubation time of 25, 30, 35, 40, 45, 50, 55, 60, 65 minutes, or higher. In some embodiments, dephosphorylation is carried out with an incubation time of 25-65 minutes, 30-60 minutes, 35-55 minutes, 40-50 minutes, 30-65 minutes, 35-65 minutes, 40-65 minutes, 45-65 minutes, 50-65 minutes, or 55-65 minutes.

[0192] In some embodiments, the differentially phosphorylated polypeptides are dephosphorylated prior to separation. In some embodiments, the differentially phosphorylated polypeptides of the disclosure are assessed relative to an appropriate reference standard (e.g., a dephosphorylated and / or unphosphorylated form of the fusion polypeptide).

[0193] In some embodiments, after separation of the differentially phosphorylated polypeptides (e.g., by ion exchange chromatography), the amount of each differentially phosphorylated polypeptide is measured. In some embodiments, the amount of each differentially phosphorylated polypeptide is measured according to various methods available in the art. In some embodiments, for example, but not limited to, the differentially phosphorylated polypeptides are measured using a malachite green assay, analytical ion exchange, spectrophotometer, colorimetric assay, and / or Western blot.

[0194] Exemplary Characterization of Metal Hydroxide Retention In some embodiments, a fusion polypeptide of the present disclosure forms a complex with a metal hydroxide (e.g., aluminum hydroxide) when exposed to it. In some embodiments, the retention of a fusion polypeptide of the present disclosure on a metal hydroxide (e.g., metal hydroxide retention) is characterized. A variety of methods are available for measuring metal hydroxide retention. In some embodiments, for example, but not limited to, metal hydroxide retention can be measured by ellipsometry, surface plasmon resonance, optical waveguide light-mode spectroscopy, attenuated total internal reflection-infrared spectroscopy, circular dichroism spectroscopy (CD), total internal reflection-infrared spectroscopy (TIRF), and other high-resolution microscopy techniques.

[0195] In some embodiments, metal hydroxide retention is characterized using an in vitro assay. For example, a known concentration of fusion polypeptide is mixed with an excess of metal hydroxide. The concentration of free, uncomplexed fusion polypeptide is quantified and compared to a standard curve to determine metal hydroxide retention. The concentration of free, uncomplexed fusion polypeptide can be assessed according to various methods known to those skilled in the art. For example, but not limited to, in some embodiments, free, uncomplexed fusion polypeptide is quantified by enzyme-linked immunosorbent assay (ELISA), Western blot, bicinchoninic acid assay, or Bradford assay.

[0196] In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the fusion polypeptide forms a complex with (e.g., is retained by) the metal hydroxide when mixed with it.

[0197] Exemplary characterization of signaling activity In some embodiments, the fusion polypeptides (and / or conjugates thereof) described herein are characterized for activity (e.g., signaling activity). In some embodiments, activity is characterized by assessing signaling (e.g., signaling capacity) in comparison to a suitable reference standard. A suitable reference standard can be, for example, a wild-type polypeptide and / or a fusion polypeptide lacking a metal hydroxide-binding polypeptide.

[0198] A variety of methods are available for assessing signaling capacity. In some embodiments, for example, signaling capacity is assessed using in vitro or in vivo-based activity assays.

[0199] In some embodiments, signaling activity is assessed using an in vitro activity assay. In some embodiments, the in vitro activity assay involves measuring activation or inhibition of downstream signaling of the fusion polypeptide. In some embodiments, measuring activation or inhibition of downstream activity involves the use of a reporter (e.g., a reporter assay). In some embodiments, the reporter assay measures activity using a detectable molecule (e.g., a reporter) that correlates with fusion polypeptide activity.

[0200] In some embodiments, reporters include fluorescent, bioluminescent, and / or other detectable probes known to those of skill in the art. In some embodiments, reporters include the use of genetic reporters. Genetic reporters can be activated, for example, based on signal transduction induced from a polypeptide. For example, detectable products or enzymes can be utilized that can be activated by the addition of a substrate to generate a detectable product and / or by-product upon activation of genetic reporter transcription. In some embodiments, enzymes useful in accordance with reporter assays are, for example, luciferase or alkaline phosphatase (e.g., secreted alkaline phosphatase, SEAP). In some such embodiments, a HEK-blue-IL12 reporter assay is utilized.

[0201] In some embodiments, signaling activity is assessed using an in vivo activity assay. In some embodiments, the fusion polypeptide is administered to a subject (e.g., a mouse, a non-human primate, a human, etc.) and activity is assessed. In some embodiments, activity is assessed by measuring activation or inhibition of downstream signaling of the fusion polypeptide, for example, compared to an appropriate reference standard (e.g., the activity of the wild-type polypeptide). Various methods are available for measuring activation or inhibition of downstream signaling of the fusion polypeptide. For example, but not limited to, alterations in differential gene expression, protein expression, and / or post-translational modifications induced by the fusion polypeptide may be measured.

[0202] Exemplary Efficacy Characterization In some embodiments, efficacy can be characterized according to one or more of a variety of available methods. In some embodiments, for example, a fusion polypeptide (or complex thereof) described herein is administered (by intratumoral or peritumoral injection) to a subject (e.g., a mouse, a non-human primate, a human, etc.), and efficacy is determined in comparison to an appropriate reference standard. A suitable reference standard can be, for example, a wild-type polypeptide and / or a polypeptide lacking a metal hydroxide-binding polypeptide or having a metal hydroxide-binding polypeptide in an unbound (e.g., unphosphorylated) state.

[0203] In some embodiments, efficacy is determined preclinically in an animal model (e.g., mouse, rat, non-human primate, etc.). In some embodiments, the fusion polypeptide is administered to the animal model (e.g., by intratumoral or peritumoral injection). For example, in some embodiments, the animal model is a tumor-bearing animal model (e.g., an animal model of cancer). In some embodiments, the cancer animal model is generated by inoculating the animal model with tumor cells. In some embodiments, the animal model is inoculated with tumor cells in the flank region. In some embodiments, the animal model is inoculated with tumor cells in a clinically relevant region (e.g., the mammary fat pad).

[0204] In some embodiments, an animal model of cancer is administered a fusion polypeptide of the present disclosure (e.g., a preparation thereof, e.g., a preparation of a phosphorylated fusion polypeptide). In some embodiments, an animal model of cancer is administered a reference standard (e.g., a wild-type polypeptide and / or a polypeptide lacking a metal hydroxide-binding polypeptide). In some embodiments, one or more of various available pre-determined measures of efficacy known in the art, such as tumor volume and / or survival rate, are assessed over time relative to a suitable reference standard (e.g., a wild-type polypeptide and / or a polypeptide lacking a metal hydroxide-binding polypeptide).

[0205] In some embodiments, the efficacy of a fusion polypeptide (e.g., a preparation thereof, e.g., a preparation of a phosphorylated fusion polypeptide) is determined clinically. In some embodiments, the fusion polypeptide is administered to a tumor-bearing subject (e.g., by intratumoral, peritumoral injection, or to a tumor-draining lymph node). In some embodiments, various available pre-determined measures of efficacy known in the art, e.g., tumor volume and / or survival rate, are assessed over time compared to tumor-bearing subjects administered a reference standard (a treatment known in the art of known efficacy and / or placebo).

[0206] Formation and Preparation of Fusion Polypeptide-Metal Hydroxide Complexes In some embodiments, phosphorylated forms of the fusion polypeptides described herein form complexes with metal hydroxides (e.g., aluminum hydroxide) when exposed to them. In some embodiments, the fusion polypeptides contain hydroxyl substituents (e.g., phosphate groups) for adsorption to metal hydroxides via ligand exchange. In some embodiments, the fusion polypeptides can form complexes with metal hydroxides via electrostatic interactions.

[0207] In some embodiments, the phosphorylated form of a fusion polypeptide comprising an IL-12 immunomodulatory domain described herein forms a complex with aluminum hydroxide when exposed to it, thus forming an IL-12 complex according to the present disclosure.

[0208] In some embodiments, a fusion polypeptide-metal hydroxide complex (e.g., an IL-12 complex) of the present disclosure is formed by mixing. In some embodiments, the mixing is performed in a buffer (e.g., a tris-buffered saline buffer). In some such embodiments, the buffer does not contain phosphate. In some such embodiments, the buffer does not contain a substance(s) that solubilize the metal hydroxide (e.g., citrate, malate, or lactate). Without wishing to be bound by any theory, buffers that contain phosphate may compete with and hinder complex formation. In some embodiments, the mixing is performed at a particular temperature for a period of time. In some such embodiments, the period of time is 5, 10, 15, 20, 25, 30, 35, 40, or 45 minutes. In some such embodiments, the specified temperature is approximately 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, or 35°C.

[0209] In some embodiments, the present disclosure provides, inter alia, a fusion polypeptide-metal hydroxide complex preparation comprising a fusion polypeptide-metal hydroxide complex according to the present disclosure. In some embodiments, the preparation comprising a fusion polypeptide-metal hydroxide complex comprises any of a variety of suitable metal hydroxides known in the art.

[0210] In some embodiments, the present disclosure provides, inter alia, pharmaceutical compositions comprising the fusion polypeptides disclosed herein. In some embodiments, the pharmaceutical compositions are formulated as fusion polypeptide-metal hydroxide complexes. In some embodiments, the pharmaceutical compositions comprise a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative, and / or adjuvant.

[0211] [Table 1-1]

[0212] [Table 1-2]

[0213] [Table 1-3]

[0214] [Table 1-4]

[0215] [Table 2-1]

[0216] [Table 2-2]

[0217] [Table 2-3] [Example]

[0218] Example 1: Optimization of the production of exemplary fusion polypeptides This example demonstrates that stable pools of cells transfected with a leap-in transposase construct containing nucleotides encoding an IL-12 fusion polypeptide and nucleotides encoding hFAM20C can be established and used to produce IL-12 fusion polypeptides. This example further demonstrates that stable pools of transfected cells are capable of producing large amounts of IL-12 fusion polypeptides.

[0219] Leap-in transposase The transposon system used in this example is designed to efficiently and consistently deliver an IL-12 fusion polypeptide and hFAM20C into host cells (e.g., CHO cells). The transposon system comprises a transposon construct (DNA) and an associated transposon enzyme (mRNA) (also called a leap-in enzyme or transposase). The transposon construct was generated by synthesizing nucleotide sequences encoding the IL-12 fusion polypeptide and hFAM20C and inserting them into a dual open reading frame (ORF) expression construct. The transposon construct was also designed to express and deliver glutamine synthase (GS).

[0220] Transposases, belonging to the DDE / D integrase family, catalyze an efficient cut-and-paste (excision followed by integration) process to integrate their associated transposons into target genomes. The enzymatic mechanism of action results in single-copy elements at multiple genomic loci. This homologous integration mechanism is several orders of magnitude more efficient than non-homologous recombination. Furthermore, it eliminates the formation of concatemers as well as deleted and rearranged transgenes. Leap-in-mediated transposition enriches integration sites in transcriptionally active genomic regions.

[0221] One of the notable features of the leap-in transposase-mediated stable integration mechanism is the strong similarity between leap-in-mediated stable pools and the monoclonal cell lines derived from those pools, both in terms of productivity and in terms of basic overall physicochemical attributes.

[0222] To ensure that the integration event is irreversible and that the transgene can be stably integrated, the transposase enzyme is delivered as mRNA. Approximately two days after cells are transfected with the mRNA, the enzyme is no longer detectable, suggesting that further transgene excision / reintegration cannot occur. In-house long-term (>3 months) experiments have shown that there is no native internal enzyme activity present in the host CHO cells that could mobilize the integrated transgene by transposition. Finally, the integration enzyme (transposase) could not be detected in a master cell bank established from a clinical manufacturing cell line developed using the leap-in system.

[0223] The transposase mRNA production process utilizes recombinant protein components but does not contain animal-derived materials.

[0224] CHO cell line A glutamine synthetase (GS)-null Chinese hamster ovary (CHO) cell line (GS-CHO cell line) was developed and used as the parent cell line (production cell line). The glutamine synthetase (GS) gene is knocked out so that the resulting cells are unable to synthesize their own glutamine and are completely dependent on glutamine supplements for growth. GS is present in the transposon construct used for transfection so that successful transfectants can be selected in glutamine-free medium.

[0225] IL-12 fusion protein:Fam20C ratio study Experiments were performed to establish the ratio of IL-12 fusion protein:Fam20C for optimal cell growth and high productivity of the fusion polypeptide.

[0226] 2:1, 4:1 and 8:1 stable pooled transfections were generated, see Table 3.

[0227] [Table 3]

[0228] Endpoint viability was >90% for all pools except for 5630 P2.2 (repeat), which dropped to 80% (see Figure 3A). Peak viable cell densities (VCDs) ranged from 18 to 27e6 / mL (Figure 3B). The lowest viability was observed for 5630 P2 (2:1 ratio). The highest viability was observed for 5631 P3 (8:1) and 5631 P4 (4:1). Overall, 5631 P3 demonstrated optimal cell growth based on a combination of viability and viable cell density (VCD). Some cell lines may achieve a higher VCD but die earlier, which is not ideal.

[0229] Cellular IL-12 fusion polypeptide production was measured by IL-12 Octet assay, and the results are shown in Table 4.

[0230] [Table 4]

[0231] The highest productivity was observed with 5631 P3 (8:1 ratio). When performed by SDS-PAGE analysis, a clear product band was observed with 5631 P3 (production run #8823) (Figure 3C).

[0232] In addition to transfections at 2:1, 4:1 and 8:1, six pools were generated at ratios of 15:1 and 8:1. See Table 5.

[0233] [Table 5]

[0234] The viability and viable cell density (VCD) are shown in Figure 4A and B. All six cell pools showed viabilities >85% and peak VCDs ranging from 15 to 23e6 / mL. Both constructs demonstrate good cell growth.

[0235] Productivity was measured by IL-12 Octet assay and is shown in Figure 5. Stable cell pools produced 50 mg / L to approximately 900 mg / L of fusion polypeptide. Pool 11370 (D208_8:1) showed higher productivity than pool 11373 (D208_15:1). This indicates that cell lines transfected at an 8:1 ratio (IL-12 fusion polypeptide:Fam20C kinase) provide stable cell lines with good viability and high IL-12 fusion polypeptide production.

[0236] Example 2: Generation of stable pools based on an 8:1 ratio This example demonstrates that stable pools of transfected cells expressing a fusion polypeptide and a kinase at a ratio of approximately 8:1 are capable of producing large amounts of IL-12 fusion polypeptide. Furthermore, these cells are capable of producing fusion polypeptides containing phosphorylated forms of the fusion polypeptide.

[0237] The GS-CHO cells and leap-in transposon system described in Example 1 are used in Example 2.

[0238] The DNA sequences of the IL-12 fusion polypeptide and hFAM20C in the transposon construct were confirmed by Sanger sequencing, and the DNA sequence of the entire stable expression plasmid was confirmed by Oxford nanopore sequencing.

[0239] Transfection Untransfected GS-CHO cells were maintained in shake flasks with growth medium and kept in a shaker incubator at 5% CO , 37°C, and 70-80% relative humidity.

[0240] Transfection of the transposon system (transposon construct and transposase mRNA) was performed using a Thermo Fisher Neon electroporation device. Post-transfection recovery was performed for 24-48 hours in growth medium in T25 flasks under static conditions. After the recovery period, transfectant cultures were placed under metabolic selection by switching the cells to a glutamine-free formulation.

[0241] Fed-batch generation and fed-batch run Fed-batch production runs were performed for pool grading in a TubeSpin bioreactor. The working volume was 10 mL and the shaker speed was 240 rpm.

[0242] On day 0 of a 14-day fed-batch run, 0.75 x 10 6 Cells were seeded at 10–15 × 10 cells / mL. On day 0, fed-batch cultures were not supplemented with additional glutamine. Cultures were maintained at 37°C, 5% CO2, and 70–80% relative humidity. Cultures were grown at 10–15 × 10 cells / mL. 6 When a density of 100 cells / mL was reached, the culture temperature was reduced to 32°C for the remainder of the run. Production runs were harvested on day 14, or earlier if viability decreased to 75%. Cell counts and metabolite measurements were taken on days 4, 7, 10, 12, and 14. Up to 12 g / L of glucose (depending on consumption rate) was added to the cultures on refeeding days using a sterile 45% glucose solution. pH was not adjusted during small-scale production runs.

[0243] Harvesting and culture clarification Harvested material from production cultures was clarified by centrifugation (3000 g, 20-30 min) and then filtered through a 0.22 μm membrane.

[0244] analysis SDA Page Method: For reducing SDS-PAGE gels, cleared supernatants were prepared in NuPAGE™ LDS Sample Buffer (4X) (Thermo Fisher catalog no. NP0008) and reduced using NuPAGE™ Sample Reducing Agent (10X) (catalog no. NP0009) according to the vendor's protocol. Reduced samples were run on NuPAGE Novex 4-12 Bis-Tris Protein Gels (catalog no. NP0329BOX, WG1403BX10) and stained using InstantBlue™ Protein Stain (Novus Biologicals catalog no. ISB1L-1L) according to the vendor's protocol. For non-reducing SDS-PAGE gels, cleared supernatants were prepared in NuPAGE™ LDS Sample Buffer (4X) (catalog no. NP0008) according to the vendor's protocol. 10 mM N-ethylmaleimide (NEM) (MilliporeSigma catalog no. E3876-5G) was added to the samples to prevent reformation of disulfide bonds. Samples were run on NuPAGE Novex 4-12 Bis-Tris Protein Gels (catalog no. NP0329BOX, WG1403BX10) and stained using InstantBlue™ Protein Stain (catalog no. ISB1L-1L) according to the vendor's protocol.

[0245] Results and Discussion Pool survival rate GS-CHO cells were transfected with an expression construct expressing an IL-12 fusion polypeptide and Fam20C kinase to generate a transfected cell pool (6880 P1) that produces the IL-12 fusion polypeptide and hFAM20C, which phosphorylates the IL-12 fusion polypeptide. An 8:1 ratio of IL-12 fusion polypeptide:Fam20C was used. After a 2-day recovery period following transfection, the cells were cultured in selective medium at 0.3 x 10 6Cells were seeded at 1000 cells / mL. This selection pressure was maintained throughout the selection period. Viable cell density and viability were determined 2-3 times weekly during selection. Viability during selection is graphed in Figure 6. Figure 6 shows a common decrease in cell viability after transfection, but importantly, the cell lines exhibit good recovery and long-term high viability (e.g., from approximately days 18 to 43 of culture). In summary, healthy, stable cells were generated when transfected with a construct having an 8:1 ratio of IL-12 fusion polypeptide:Fam20C.

[0246] Cell productivity and quality of fusion polypeptide To estimate productivity and protein quality, a fed-batch production run was initiated from a 10 mL culture in TubeSpins for cell pool 6880 P1. Productivity was measured over the course of the fed-batch run by biolayer interferometry (BLI). Productivity and product quality were also assessed using SDS-PAGE. The clarified harvest was diluted using sample diluent (Pall ForteBio catalog no. 18-1048) and measured by biolayer interferometry (BLI) in 96-well plates on an Octet HTX instrument using a Protein A sensor. Appropriate sample dilutions were generated, and all valid measurements were within the standard concentration range. Multiple replicates were also performed to reduce dilution- and sensor-dependent measurement error. IL12 samples were measured on an Octet HTX instrument (ForteBio) using the standard protocol. Mouse anti-human IL12 antibody (BioLegend catalog no. 508808) was loaded onto an anti-mouse Fc capture chip (AMC, ForteBio). IL12 fusion polypeptide concentrations were calculated in ForteBio Data Analysis HT 11.1 by measuring the observed binding rate using a four-parameter logistic (4PL) curve compared to a standard curve using purified IL-12 (protein version ID 67074.1.a) ranging from 1.56 to 100 μg / ml. The clarified supernatant was diluted in 1X Kinetics Buffer (ForteBio) so that the measured concentration was within the range of the standard curve. Additional qualitative analysis was performed by SDS-PAGE. Performance parameters for Pool 6880 P1 during the fed-batch production evaluation are shown in Figure 7A-C.

[0247] The transfected cells showed greater than 90% viability during the 14-day fed-batch run (Figure 7A), and they were able to produce 100 mg / L (day 7) to 250 mg / L (day 14) of IL-12 fusion polypeptide (Figure 7C).

[0248] Malachite green assay of purified IL-12 fusion polypeptide from the day 14 harvest of pool 11370. IL-12 fusion polypeptide was purified using a first anion exchange chromatography step and a hydrophobic interaction chromatography step. This purification process did not include a second anion exchange polishing step. IL-12 fusion polypeptide demonstrated approximately 5 phosphates per fusion polypeptide.

[0249] Example 3: Bioreactor production of IL-12 fusion polypeptides The transfected GS-CHO cells from Example 2 were further cultured and tested.

[0250] Only GS-CHO cells expressing detectable levels of IL-12 fusion polypeptide were sorted into 96-well plates at one cell per well using a fluorescence-activated cell sorter. In total, approximately 5700 wells were targeted for seeding with a single cell.

[0251] A total of 644 wells were identified as containing growing cells. From these wells, 539 cell lines were screened for product expression; 122 colonies expressed detectable levels of product. A total of 80 highly ranked cell lines were transferred to suspension culture.

[0252] After transfer to suspension culture, the growth and productivity of 24 highly ranked cell lines were evaluated in a miniature bioreactor system (ambr® 250 system; TAP Biosystems / Sartorius Stedim Biotech). Selection of the 24 cell lines was based on static productivity evaluation of the cell lines and acceptable growth (1.0 × 10 6The ranking was based on IL-12 fusion polypeptide product concentration, assessed by a viable cell concentration (VCC) on the day of subculture that consistently exceeded 100 cells / mL and cell viability greater than 90%. Twenty-three cell lines were successfully evaluated. IL-12 fusion polypeptide concentrations at harvest ranged from 189.1 to 742.8 mg / L, as determined by Octet analysis.

[0253] High productivity (e.g., at least 180 mg / mL) in fed-batch miniature bioreactor evaluations and acceptable growth characteristics during routine subculture in shake flask cultures (e.g., 1.0 × 10 cells / mL upon subculture). 6 Eight cell lines were selected for further evaluation based on a cell density consistently exceeding 10 ...

[0254] [Table 6]

[0255] A 12-vial research cell bank of each of the eight selected lead candidate cell lines was cryopreserved.

[0256] Products from harvested clarified conditioned medium (CCS) from FMB1 cultures of eight lead candidate cell lines were purified by CH1 affinity purification using the AKTA system (Section 4.17). Partially purified supernatants were characterized using UPLC-MS for N-linked oligosaccharide analysis, GP HPLC for aggregate / fragment analysis, PMAP-MS for N-linked and O-linked oligosaccharide analysis, ESI-MS for intact protein structural characterization, malachite green for phosphorylation analysis, RP HPLC for sialic acid determination, and RP UPLC for purity determination.

[0257] Data analysis Protein Concentration Determination Using the Octet® System The Octet® HTX System (ForteBio Biologics / Molecular Devices LLC) was used to measure biolayer interferometry of CCS samples to allow grading by product concentration. The system uses a glass fiber biosensor coated with a special optical layer and a capture molecule bound to the tip. The tip is immersed in a sample containing target molecules. The target molecules bind to the capture molecules, and the two form a molecular layer. When white light is directed at the biosensor, two beams are reflected back: 1. from the tip as a reference, and 2. from the molecular layer. Interference between the two beams causes a wavelength shift, which depends on the thickness of the molecular layer and corresponds to the number of molecules on the tip surface. As target molecules bind to the biosensor, the wavelength shift, expressed in nm, is plotted in real time to generate a binding curve. Concentration-dependent binding curves were obtained for standards, controls, and samples, representing the rate of increase in molecular layer thickness as product bound to capture molecules on the biosensor tip surface under constant conditions. The binding rate was determined from the binding curves using Octet® Data Analysis software. The binding rates of standards of known concentrations were plotted as a standard curve and used to estimate the product concentrations in the samples and rank them accordingly.

[0258] Hydroxyapatite type 1 resin (CH1) affinity purification using AKTA Product purification for analytical purposes only was performed using CaptureSelect CH1-XL with preset binding and elution conditions. As the product-containing solution was passed through the resin, the molecule of interest reversibly bound to the CH1 affinity ligand, flushing impurities through the column. The bound product was then recovered by lowering the pH with an elution buffer, which denatures the resin-product bond. Thus, the product could be separated from contaminants. After purification, the sample was neutralized and ready for analysis of product quality. The eluate concentration of was measured using the Nanodrop system.

[0259] UPLC-MS analysis The UPLC-MS high-throughput N-glycan analysis platform involved high-throughput glycan preparation using the GlykoPrep Rapid 2-AB Kit with the AssayMAP Bravo Liquid Handler, followed by UPLC and mass spectrometry analysis. The sample preparation workflow consisted of automated purification and normalization steps, followed by digestion with the enzyme peptide-N-glycosidase F to release N-glycans, their separation from glycoproteins, fluorescent labeling with the fluorophore 2-aminobenzamide (2-AB), and cleanup for analysis. The labeled glycans were analyzed by hydrophilic interaction UPLC coupled to electrospray time-of-flight mass spectrometry. Analysis was performed using an AQUITY UPLC H-Class Bio System and an AQUITY UPLC Fluorescence Detector in series with a Xevo G2S Q-TOF system operated in sensitivity and positive ionization modes.

[0260] Quantification and identification of oligosaccharide structures were performed using the Glycan Workflow in UNIFI 1.8.2 software. A 2-AB-labeled dextran ladder was used to calibrate and normalize 2-AB-labeled glycan retention times to glucose units. Initial assignments of oligosaccharide species for neutral and charged oligosaccharide profiling were made based on comparison of glucose units to the NIBRT glycan database. These initial assignments were confirmed by mass spectrometry. The percentage of each glycan was based on the area of ​​each peak relative to the total integrated peak area. Using this method, some minor abundance peaks may remain unidentified, even though they have similar glucose unit values ​​to entries in the NIBRT glycan database, because their proportions are too low to be detected by mass spectrometry. For this reason, peaks below 0.3% are not reported.

[0261] Gel permeation (GP) HPLC analysis GP HPLC was used to separate the product monomer from both aggregates and fragments. Monomeric components were identified by their characteristic retention time and location relative to calibration markers. Aggregate analysis was performed using a TSKgel® G3000SWXL column (Hichrom Ltd). Product components were detected by measuring A280 nm, and peak chromatograms were analyzed using Empower™ 2 software (Waters Corporation). The proportions of sample components were determined by calculating the peak area of ​​each component relative to the total integrated peak, which was used to measure the product concentration in the sample.

[0262] Peptide mapping mass spectrometry (PMAP-MS) analysis PMAP-MS analysis was performed using a Waters Acquity UPLC and a Waters XevoG2 QTO. Samples underwent trypsin digestion. The MS was calibrated for mass assignment, resolution, and sensitivity prior to analysis. A Glu-fibrinogen lock spray was acquired every 30 seconds as a reference scan. Data analysis was performed using MassLynx and BiopharmaLynx software. The focus of this PMAP-MS analysis was comparison of peaks within the total ion chromatogram (TIC). MS1 and MS / MS data were acquired for all samples. Only MS1 data was used for analysis; MS / MS datasets provided additional information as needed. The following were not covered in this analysis: sequence coverage estimation, clipping, pyroglutamation, deamidation, glycation, and oxidation.

[0263] Electrospray ionization mass spectrometry (ESI-MS) analysis ESI-MS analysis was performed using a Waters Acquity UPLC and a Waters XevoG2 QTOF. Samples were deglycosylated and reduced according to UKSL-1795 without buffer exchange for sample restriction (the cloned buffer components were compatible with this method). LCMS analysis was performed using a XevoG2 QTOF mass spectrometer operating in positive ion mode over the m / z range of 600 to 4000. Data analysis was performed using Protein Metrics software (version 3.9) utilizing a parsimonious deconvolution algorithm. Appropriate processing parameters were selected for the analyzed products, and assigned masses were reported from this processing.

[0264] Malachite Green The degree of phosphorylation of purified IL-12 fusion polypeptides was determined using the Thermo Scientific™ Phosphoprotein Phosphate Estimation Assay Kit. The kit utilizes alkaline hydrolysis of phosphate groups from seryl and threonyl residues in phosphoproteins. The liberated phosphate is complexed with ammonium molybdate upon addition. Malachite green reagent then forms a secondary complex with phosphomolybdate, resulting in a color change reaction. The absorbance of the samples was measured using a microplate reader and extrapolated against a five-point standard curve of known phosphate concentrations.

[0265] Sialic acid determination Sialic acid determination in test samples was performed by RP-HPLC. The sample preparation workflow consisted of dilution of the test sample followed by hydrolysis using TFA solution. The hydrolyzed test sample was then labeled with DMB solution to form a fluorescent reaction product. Quantification of N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) was performed against external standards using RP-HPLC with fluorescence detection. Samples were analyzed by reversed-phase (RP)-HPLC using an Agilent 1100 / 1200 series HPLC system.

[0266] Reversed-phase ultra-performance liquid chromatography (RP-UPLC) analysis RP-UPLC with fluorescence detection (FLD) is used to quantify the relative percent total purity of the IL-12 fusion polypeptide product. Reverse-phase chromatography separates sample components based on differences in hydrophobicity. The sample is injected onto a column (stationary phase), and interactions with the hydrophobic surface remove them from the mobile phase. Sample components bind to the column depending on the strength and nature of their hydrophobic and polar interactions. The product is eluted by increasing the organic strength of the mobile phase. Less hydrophobic components elute earlier, and more hydrophobic components elute later. The eluted components are monitored using fluorescence.

[0267] result The IL-12 fusion polypeptides produced by all eight cell lines (NKB05, NKB19, NKB23, NKB28, NKB44, NKB46, NKB70, and NKB78) were similar when analyzed for aggregates by GP HPLC, phosphorylation by malachite green, and sialylation by RP-HPLC. Some differences were observed in the proteins produced by these cells when analyzed for oligosaccharides by UPLC-MS and major phosphorylation sites by PMAP-MS, which were not sufficient to exclude any of the cell lines from further evaluation.

[0268] [Table 7]

[0269] Finally, differences were observed in the intact protein characterization data for two of the cell lines that indicated product fragmentation in these cell lines.

[0270] Characterization data for lead cell lines

[0271] [Table 8]

[0272] [Table 9]

[0273] [Table 10]

[0274] Following fed-batch miniature bioreactor evaluation, cell line NKB46 was selected as the tentative lead cell line for preparation of a master cell bank and future cGMP manufacturing of the IL-12 fusion polypeptide. This selection was based on product characteristics, including high productivity in fed-batch miniature bioreactor evaluation, high specific production rate, and acceptable growth and degree of phosphorylation from fed-batch miniature bioreactor evaluation. This cell line was renamed INTRA2021 for current Good Manufacturing Practice (cGMP) purposes.

[0275] Example 4: Exemplary Purification Scheme This example demonstrates an exemplary method for purifying IL-12 fusion polypeptides from transfected cells.

[0276] Exemplary purification methods include: Depth filtration or centrifugation to harvest the bioreactor (e.g., typically a 2-week fed-batch run, as in Examples 2 or 3) to remove cells and cell debris · Viral inactivation with low pH or detergents for CHO-based processes.

[0277] a first chromatography step, e.g., a capture step (e.g., an anion exchange capture chromatography step); A second chromatography step, e.g., a polishing step (e.g., hydrophobic interaction chromatography) A third chromatography step, e.g., a final polishing step (e.g., an anion exchange capture chromatography step). UF / DF for formulating bulk drugs in appropriate buffers and excipients Example 6: Conventional Capture Chromatography Equilibrate the resin / column with an appropriate buffer to adjust the column to the appropriate pH (e.g., 6.8-7.4) and conductivity (salt, e.g., NaCl). Load the column with the appropriate load (typically clarified cell media matching the pH and conductivity of the equilibration buffer). Wash the load with equilibration buffer until all of the load material has been washed from the column (A280 drops to baseline). Another wash is performed before eluting the product (IL-12 fusion polypeptide) to remove impurities. Elute the phosphorylated fusion polypeptide by either increasing / decreasing the salt concentration or changing the pH. Strip the column from any remaining bound material with high / low salt, chaotropes, alcohol, or NaOH.

[0278] Example 7: Purification-1 This example demonstrates that a first anion exchange chromatography (AEX) step followed by a second hydrophobic interaction chromatography step can be used to separate the phosphorylated form of a fusion polypeptide from lower and higher molecular weight impurities (e.g., aggregated IL-12 fusion polypeptide) as well as host cell impurities, and that a highly pure preparation of the phosphorylated form of the fusion polypeptide can be obtained.

[0279] In this example, a first anion chromatography (AEX) step is used to capture the IL-12 fusion polypeptide from the cell extract, followed by either 1) a hydroxyapatite chromatography capture step or 2) a hydrophobic interaction refinement chromatography step as a subsequent refinement step.

[0280] Culture Harvest and Clarification Fed-batch harvesting was performed on day 14 and clarified by centrifugation. 200 mL aliquots were stored frozen. Aliquots were thawed in a room temperature bath, and clarification was performed by secondary depth filtration. The filters were flushed with DI H2O and equilibrated with TBS (20 mM Tris pH 7.4, 150 mM NaCl) before feasibility testing.

[0281] Anion Chromatography Capture Step (AEX) An exemplary method for anion exchange capture chromatography is shown below.

[0282] Column: Tosoh GigaCap Q-650M, 5 mL, 1.46 cm D x 3 cm H Equilibration buffer (A): 20mM Tris pH7.4 Elution buffer (B): 20mM Tris pH7.4 + 1M NaCl Purification buffer: 0.5N NaOH Load preparation: The clarified harvest was diluted 1:1 (v:v) with DI H2O.

[0283] The eluted fractions were stored at 4°C overnight.

[0284] [Table 11]

[0285] Figure 8 shows the full chromatogram and zoom of the elution peak during the first chromatography of the clarified harvest of stable pool 6880 P1 by anion exchange chromatography (GigaCap Q). The SDS-PAGE gel demonstrates that the IL-12 fusion polypeptide (band approximately 75,000 daltons) was captured by the resin, demonstrating that anion exchange capture chromatography can be used to separate the IL-12 fusion polypeptide from lower and higher molecular weight impurities (Figure 9).

[0286] Hydroxyapatite chromatographic capture step The IL-12 fusion polypeptide was first captured by AEX Gigacap Q (5 mL) and then purified (refined) by a refined hydroxyapatite capture chromatography step. An exemplary method for hydroxyapatite capture chromatography is included below.

[0287] Column: BioRad CHT XT, 1 mL, 0.8 cm D x 2 cm H EQ buffer (A): 5mM NaPO4 pH6.8, 20ppm Ca++, 50mM NaCl, 50mM MES Elution buffer (B): 500mM NaPO4 pH6.8, 20ppm Ca++, 50mM NaCl, 50mM MES Regeneration buffer: 400mM NaPO4 pH7.0 Purification buffer: 1N NaOH Load preparation: The XT pool was diluted 1:2 (v:v) with EQ buffer, 0.5x volume per load (2 column runs were performed).

[0288] [Table 12]

[0289] Figure 10 shows the full chromatogram and zoom of the elution peak during the first chromatography of the clarified harvest of stable pool 6880 P1 with hydroxyapatite chromatography as the second chromatography step. The SDS-PAGE gel demonstrates that hydroxyapatite chromatography can be used to separate the IL-12 fusion polypeptide from lower and higher molecular weight impurities (Figure 11). The majority of the product elutes in fraction A, while fraction B contains significant amounts of lower molecular weight impurities.

[0290] Hydrophobic interaction refinement chromatography step The IL-12 fusion polypeptide was first captured by AEX Gigacap Q (5 mL) and then purified (polished) by either heparin HP or phenyl HP chromatography.

[0291] [Table 13]

[0292] Non-reducing SDS-PAGE was loaded at 3 μg / lane (FIG. 12). An example of a comparison of phenyl chromatography (hydrophobic interaction) to hydroxyapatite (CHT) after initial capture with AEX chromatography is shown in FIG.

[0293] The phenyl fraction eluted with 1 M salt (NH4SO4) (lanes 8-10, indicated by circles) demonstrates high IL-12 fusion polypeptide purity when compared to the CHT elution fraction (lanes 3-4), indicating that IL-12 fusion proteins can be isolated with high purity.

[0294] An exemplary method for Phenyl HP chromatography is included below.

[0295] Column: Phenyl Sepharose HP (GE 17-1082-03) 5 mL, 1 cm D x 6.4 cm H EQ buffer (A): 20mM Tris pH7.4, 1.5M ammonium sulfate Elution buffer (B): 20mM Tris pH7.4 Wash 2 buffer: EQ buffer + 5% isopropyl alcohol Purification buffer: 0.01N NaOH Load preparation: Add 7.5 mL of 4M 1.5M ammonium sulfate and clarify by centrifugation The eluted fractions were stored at 4°C overnight.

[0296] [Table 14]

[0297] Analysis of the fractions eluted from the Phenyl HP column demonstrates the high purity of the IL12 fusion molecule as assessed by SDS-PAGE and SEC-HPLC analysis (Figure 13).

[0298] Another example of phenyl chromatography where more refined conditions of loading and elution produced highly pure material (phenyl chromatography after AEX capture step).

[0299] Example 8: Purification-2 This example demonstrates that phenyl chromatography reduces impurities and Q chromatography separates product from aggregated products, allowing isolation of specific IL-12 fusion polypeptide phosphorylation levels.

[0300] Cells were harvested and clarified as in Example 7.

[0301] Affinity Chromatography Capture Step An exemplary method for affinity capture chromatography is shown below.

[0302] Column: Cytiva Heparin Sepharose 6 FF, 45 mL (2.6 cm D x 8.5 cm H) Equilibration buffer (A): 20mM Tris pH7.4 Elution buffer (B): 20mM Tris pH7.4 + 2M NaCl Purification buffer: 0.1N NaOH Load preparation: The clarified harvest was diluted 1:1 (v:v) with equilibration buffer.

[0303] [Table 15]

[0304] Example of affinity capture chromatography with heparin sepharose where the elution conditions demonstrated separation between the IL12 fusion polypeptide and lower molecular weight impurities as revealed by SDS-PAGE analysis (Figure 14).

[0305] Hydrophobic interaction refinement chromatography step An exemplary method for Phenyl HP chromatography is included below.

[0306] Column: Phenyl Sepharose HP (GE 17-1082-03) 11 mL, 1.0 cm D x 14 cm H EQ buffer (A): 20mM Tris pH7.4, 100mM NaCl, 1.5M ammonium sulfate Elution buffer (B): 20mM Tris pH7.4 Purification buffer: 0.01N NaOH Load preparation: Adjust the heparin elution pool to 1.5 M ammonium sulfate with 4.0 M ammonium sulfate stock. Let stand 30 minutes and filter the load through a 0.45 micron filter (vacuum flask).

[0307] Load 0.5x volume per run (2 runs total)

[0308] [Table 16]

[0309] Figure 14 shows a non-reducing, stain-free 4-10% SDS-PAGE gel loaded with heparin or phenyl fractions (3 µg of sample per lane). The elution fraction (lane 7) demonstrated significant purification when compared to lane 9 (phenyl chromatography load). SEC-HPLC analysis (Figure 16A) also demonstrated a higher purity of the phenyl pool than its load (heparin Sepharose eluate).

[0310] Anion Chromatography Capture Step An exemplary method for anion exchange capture chromatography is shown below.

[0311] Column: Cytiva Q Sepharose HP, 11 mL, 1.0 cm D x 14 cm H Equilibration buffer (A): 20mM Tris pH7.4 Elution buffer (B): 20mM Tris pH7.4 + 1M NaCl Purification buffer: 0.5N NaOH Load preparation: Pool both Phenyl 0.75M E1 eluates. Dilute to 0.1x in Buffer A.

[0312] The conductivity of the load is approximately 16 mS / cm.

[0313] [Table 17]

[0314] Figure 15 shows a non-reducing, stain-free 4-10% SDS-PAGE gel loaded with 3 μg of sample per lane. AEX as a final polishing step produced highly pure material (SDS-PAGE and SEC-HPLC, Figures 15 and 16A) and demonstrated the ability to separate the monomeric product from aggregates (fractions 419 and 424, Figure 16B). Malachite green assay results for fractions from Q HP chromatography are shown in Table 14.

[0315] [Table 18]

[0316] This exemplary purification method demonstrates that phenyl chromatography significantly reduces product and process impurities as revealed by SDS-PAGE. SEC-HPLC and its Q-chromatography are powerful tools for separating aggregated products (SEC-HPLC), product from other impurities, and for selecting the level of phosphorylation (Malachite Green data).

[0317] Example 9: Purification-3 This example demonstrates that a purification process using three chromatographic steps provides a highly purified IL-12 fusion polypeptide product (>98% SEC-HPLC, and >82% and >55% for non-reducing and reducing RP HPLC, respectively) with acceptably low and detergent-reduced levels of host cell proteins (HCPs), DNA, low molecular weight species (LMWS), and high molecular weight species (HMWS). This method provided an IL-12 fusion polypeptide with the desired phosphorylation level, exhibiting approximately 7-8 phosphomolecules per molecule of protein by malachite green assay.

[0318] Lab-scale run-through (LSRT) of the purification process for the product IL-12 fusion polypeptide from a pool of GS-CHO cells transfected as described in the previous examples herein.

[0319] An exemplary purification process for ANK-101 (IL-12 fusion polypeptide) includes three chromatography steps: TOYOPEARL GigaCap Q-650M anion exchange chromatography (equivalent to the Q chromatography used in Examples 7 and 8), Capto Phenyl HS hydrophobic interaction chromatography (equivalent to the phenyl chromatography used in Examples 7 and 8), and TOYOPEARL GigaCap Q-650S anion exchange chromatography (equivalent to the Q chromatography used in Examples 7 and 8).

[0320] The process also included a viral inactivation step prior to loading into the first chromatography step. The second chromatography step was followed by an intermediate ultrafiltration / diafiltration (UF / DF). The third step was followed by viral reduction filtration sizing and final dilution. These are all steps that may accompany a chromatography step in the manufacturing process.

[0321] Detailed purification workflow: TDAO virus inactivation TOYOPEARL GigaCap Q-650M Anion Exchange Chromatography (AEX) Capto Phenyl HS Hydrophobic Interaction Chromatography (HIC) Intermediate Ultrafiltration / Diafiltration (UF / DF) TOYOPEARL GigaCap Q-650S Anion Exchange Chromatography (AEX) Planova BioEX Virus Reducing Filtration (VRF) UF / DF and excipient addition The TOYOPEARL GigaCap Q-650M chromatography step was performed at a linear flow rate of 300 cm / h using the load capacity of 21.4 g / L resin determined during this step. The average step recovery determined based on the octet data was 100%, and the average step recovery determined based on the RP HPLC data was 85.7%.

[0322] The Capto Phenyl HS chromatography step was performed at a load capacity of 15.0 g / L and a linear flow rate of 275 cm / h using 20 mM Tris, 100 mM sodium chloride, 1.4 M ammonium sulfate pH 7.4 as the equilibration buffer. The IL-12 fusion polypeptide was eluted with 20 mM Tris, 100 mM sodium chloride, 740 mM ammonium sulfate pH 7.4. The average step recovery was 65.4%.

[0323] Further refinement experiments were evaluated on ceramic hydroxyapatite (CHT XT) and TOYOPEARL GigaCap Q-650S. Data obtained on TOYOPEARL GigaCap Q-650S indicated the desired product quality, low impurity levels, and high efficiency for separating phosphorylated species using an isocratic elution step. A three-chromatography step process was designed for the purification of the IL-12 fusion polypeptide. The average step recovery was 51.3%.

[0324] Planova BioEX filter sizing results suggested a 1.0 m² membrane for GMP-scale processing of material in 5 hours. For some methods, the maximum volumetric throughput within the 5-hour processing time was 262.6 L / m². The final product was diluted in 20 mM Tris, 50 mM sodium chloride, pH 7.3 to a protein concentration of 2.040 g / L. This purification step successfully recovered 1326.17 mg (83.8%). Excipients were added to the diafiltered product to achieve a final composition of 20 mM Tris, 50 mM sodium chloride, 10 mM methionine, 150 mM sucrose, 0.02% polysorbate, pH 7.3, at a protein concentration of 2.006 g / L. The estimated cumulative process yield was 19.7%.

[0325] Analytical testing of the bulk purified IL-12 fusion polypeptide product resulting from the final purification process showed that the product purity was greater than 98% as measured by GP HPLC analysis, and greater than 82% and 55% by non-reducing and reducing RP HPLC, respectively.

[0326] Impurity analysis of the bulk-purified product showed DNA and HCP levels of 0.3 pg / mg and 4.2 ng / mg, respectively. The bulk-purified IL-12 fusion polypeptide contains approximately seven to nine (e.g., seven) phosphomolecules, as determined by malachite green assay. Intact LC ESI-MS showed 6X, 7X, 8X, 9X, 10X, 11X, and 12X phosphorylation sites present in the bulk-purified IL-12 fusion polypeptide product. Intact LC ESI-MS also showed the +9 phosphate as the most predominant species for the bulk-purified product.

[0327] TOYOPEARL GigaCap Q-650M anion exchange chromatography as the first step Below are a number of exemplary methods demonstrating that anion exchange chromatography (e.g., TOYOPEARL GigaCap Q-650M) can be used as the first capture step for IL-12 fusion polypeptides. Using anion exchange chromatography as the first capture step achieves significant reduction of host cell proteins (HCPs) and higher purity. Anion exchange chromatography also enriches the preparation for highly phosphorylated species.

[0328] An exemplary method for anion exchange capture chromatography using a linear gradient elution approach is described in Table 19.

[0329] [Table 19]

[0330] [Table 20]

[0331] Based on the pooling strategy, the first capture step (anion exchange chromatography) achieves a significant reduction in host cell proteins (HCPs), mock pool 4, and higher purity.

[0332] An exemplary method for anion exchange capture chromatography using a stepwise or isocratic conditions approach is described in the table below.

[0333] [Table 21]

[0334] An exemplary method for anion exchange capture chromatography is described in the table below.

[0335] [Table 22]

[0336] [Table 23]

[0337] The results show that multiple cycles on the GigaCap Q 650M under optimized conditions result in consistent purity and recovery as revealed by eluate concentration, SEC-HPLC, and RP-HPLC under reducing and non-reducing conditions (Table 19).

[0338] Capto Phenyl HS Hydrophobic Interaction Chromatography (HIC) as a Second Step Below are a number of exemplary methods demonstrating that hydrophobic interaction chromatography (e.g., Capto Phenyl HS) can be used as a second chromatographic step (polishing step) when purifying IL-12 fusion polypeptides. Hydrophobic interaction chromatography as a second chromatographic step for IL-12 fusion polypeptides demonstrated high purity and host cell protein (HCP) removal, and demonstrated excellent performance in terms of both product recovery (approximately 55%) and purity (98.74% monomer, 89.9% NR RP-HPLC, 67% Red RP-HPLC, 92 ppm HCP).

[0339] An exemplary method for hydrophobic interaction chromatography using a linear gradient elution approach is described in the table below.

[0340] [Table 24]

[0341] An exemplary method for hydrophobic interaction chromatography using a stepwise or isocratic conditions approach is described in the table below.

[0342] [Table 25]

[0343] [Table 26]

[0344] [Table 27]

[0345] The conditions demonstrated high purity and host cell protein (HCP) removal over multiple column runs (column cycling 10 times). The conditions resulted in excellent performance in terms of both product recovery (approximately 55%) and purity (98.74% monomer, 89.9% NR RP-HPLC, 67% Red RP-HPLC, 92 ppm HCP).

[0346] An exemplary method for hydrophobic interaction chromatography is described in the table below.

[0347] [Table 28]

[0348] TOYOPEARL GigaCap Q-650S anion exchange chromatography as the third step Below are a number of exemplary methods demonstrating that anion exchange chromatography (e.g., TOYOPEARL GigaCap Q-650S) can be used as a third capture step for IL-12 fusion polypeptides. During this step, the sodium chloride concentrations in the post-load wash and elution buffers are optimized to select for optimally phosphorylated IL-12 fusion polypeptides and purify the product from aggregated species and other impurities (host cell proteins and host cell DNA). After loading the column, the unbound or flow-through material is washed with equilibration buffer (post-load wash (PLW1)), whereas PLW2 is utilized to remove less phosphorylated species. After elution, the column is stripped with high salt to remove other impurities, such as product aggregates, host cell proteins, and host cell DNA (post-elution wash or PEW).

[0349] For initial gradient evaluation, one cycle was performed on TOYOPEARL GigaCap Q-650S resin using material diafiltered into 20 mM sodium phosphate pH 7.0 generated through additional UF / DF. Cycles were performed using a 25 CV gradient of sodium chloride and a load capacity of 10.0 g / L (resin) for the elution step. The elution peak was fractionated, and fractions were analyzed by SEC-HPLC, RP HPLC, and malachite green assay. Different fractions were combined, and a mock pool was also submitted for additional impurity analysis (DNA and HCP). The analytical results are summarized in Table 29.

[0350] [Table 29]

[0351] [Table 30]

[0352] [Table 31]

[0353] [Table 32]

[0354] Chromatography on GigaCap Q-650S using stepwise or isocratic elution. PLW2 = Post Load Wash 2 (pre-elution). The goal of this wash is to remove certain impurities before elution of the product.

[0355] [Table 33]

[0356] [Table 34]

[0357] [Table 35]

[0358] [Table 36]

[0359] The final run conditions for the TOYOPEARL GigaCap Q-650S are PLW2 (post-load wash) buffer containing 274 mM sodium chloride and elution buffer containing 355 mM sodium chloride. Load volumes up to 20 g / L can be used. The elution collection criteria were set at an absorbance of A280 20 mAU to 500 mAU (A280 0.1 OD to 2.5 OD). PLW2 and elution buffers were formulated to tight buffer specifications because variations can affect the final polishing step.

[0360] Figure 17 shows multiple cycles of the GigaCap 650S column performed under optimized conditions.

[0361] [Table 37]

[0362] [Table 38]

[0363] [Table 39]

[0364] In-process samples were analyzed by Malachite Green kit assay and the results are shown below.

[0365] [Table 40]

[0366] In-process samples for each chromatography step were analyzed at Eurofins for the TDAO (an exemplary viral inactivation buffer) detergent detection assay, and the results are shown below.

[0367] [Table 41]

[0368] overview The purification process, in some manufacturing methods, may include three chromatography steps (e.g., TOYOPEARL GigaCap Q-650M anion exchange chromatography; Capto Phenyl HS hydrophobic interaction chromatography followed by TOYOPEARL GigaCap Q-650S anion exchange chromatography). Optionally, a retroviral inactivation step in the form of detergent treatment of the cell culture supernatant may be included prior to TOYOPEARL GigaCap Q-650M chromatography. Optionally, Planova BioEx virus reduction filtration may also be included after the final polishing chromatography step. Optionally, an intermediate UF / DF step may be introduced after Capto Phenyl HS to reduce conductivity and exchange buffer for the next chromatography step.

[0369] The results suggest that in one method for producing IL-12 fusion polypeptides, the load capacity of the TOYOPEARL GigaCap Q-650M anion exchange chromatography is approximately 21.4 g per L of resin at a flow rate of 300 cm / h. The results also suggest that eluate collection should be defined as beginning when A280 nm is ≥ 570 mAU on the ascending phase and ending when A280 nm is ≤ 300 mAU on the descending phase. The process yield exceeded 100%, but this may be a consequence of the titration method used, since octets were used for HCCF titration and NanoDrop assays were used to quantify the product on the eluate.

[0370] Various conditions for binding and elution were evaluated for the Capto Phenyl HS chromatography step. In some embodiments, a load capacity of 15.0 g per L of resin may be used. HCP and HMWS removal data, as well as yield data, suggested the selection of an elution buffer (20 mM Tris, 740 mM ammonium sulfate, pH 7.4) for purification of the IL-12 fusion polypeptide through the final purification process. Optionally, a clarification procedure including sodium hydroxide, WFI, and guanidine hydrochloride may be performed, as this may improve resin regeneration.

[0371] Optionally, an intermediate ultrafiltration / diafiltration can be introduced after Capto Phenyl HS chromatography to condition the material before the final refinement step.

[0372] The IL-12 fusion polypeptide was slightly concentrated and the buffer exchanged into 20 mM Tris pH 7.3. The retentate showed greater than 98% monomer, but high levels of HMWS and LMWS were observed in the buffer flush. In fact, buffer flush recovery was extremely low (less than 1% yield), so it is recommended not to include a buffer flush from the intermediate UF / DF in future purification runs until the fractions are shown to be free of significant HMWS and LMWS.

[0373] Isocratic wash and elution conditions were developed for the TOYOPEARL GigaCap Q-650S chromatography system to achieve the desired product quality. Various sodium chloride molarity concentrations in the PLW2 and elution buffer were evaluated, along with collection criteria. The final step conditions were determined using 274 mM sodium chloride in the PLW2 buffer and 355 mM sodium chloride in the elution buffer, with a loading of 20 g / L. To achieve the desired phosphorylation of the product, eluate collection was defined to begin when A280 nm was ≥ 20 mAU on the ascending phase and end when A280 nm was ≤ 500 mAU on the descending phase. It should be noted that the conductivity specifications for both the PLW2 and elution buffer were stringent and critical for performing the final polishing step.

[0374] In some embodiments, a filtration step is included in the manufacturing process. Planova BioEX filter sizing results suggested that 1.0 m2 of membrane could be used for 1000 L GMP scale to process material in 5 hours in a manufacturing facility.

[0375] In some embodiments, the TOYOPEARL GigaCap Q-650S eluate containing the IL-12 fusion polypeptide is diluted to 1-3 g / L (e.g., 2.040 g / L) in an appropriate buffer (e.g., 20 mM Tris, 355 mM sodium chloride pH 7.3) and referred to as the bulk purified product (BBP).

[0376] The overall cumulative yield of the developed downstream process was 35.1%.

[0377] GP HPLC analysis demonstrated that the IL-12 fusion polypeptide product was purified to greater than 98% in the bulk-purified product during this purification process. Non-reduced and reduced RP HPLC also demonstrated high purity, up to greater than 82% and 55%, respectively. The process also successfully reduced the levels of HCP, DNA, LMWS, and HMWS to acceptably low levels. Detergent removal throughout the purification process to acceptable levels was observed. The bulk-purified product exhibited the desired phosphorylation level, approximately seven phosphorus molecules per protein molecule by malachite green assay. Intact ESI-MS showed 5X, 6X, 7X, 8X, 9X, 10X, 11X, and 13X phosphorylated species in the final bulk product compared to the reference standard batch (C-150921-0079), which exhibited 3X, 4X, 5X, 6X, 7X, 8X, and 9X phosphorylated mass species. Fragmentation at 5X, 7X, 9X, 11X and 13X phosphorylated species was also observed in the final bulk product.

[0378] Example 10: Exemplary 500 L and 1000 L Scale Production This example demonstrates the manufacture of two batches of IL-12 fusion polypeptide drug substance (a 500 L reference batch and a 1000 L current good manufacturing practice (GMP) batch). Both batches produce IL-12 fusion polypeptide drug substance of comparable high quality. The IL-12 fusion polypeptide drug substance exhibited high purity (e.g., less fragmentation and / or less aggregation), high heterogeneity, favorable phosphorylation, and / or high activity.

[0379] manufacturing an IL-12 fusion polypeptide drug substance, 500L scale bioreactor batch (analytical reference standard batch) (P4130826ARS); and 1000L scale bioreactor batch (Good Manufacturing Practice (GMP) batch) (1205114) Manufactured in.

[0380] A 500 L scale bioreactor batch (P4130826ARS) was produced using multiple rounds of bind-and-elute chromatography (including a first anion chromatography step, a hydrophobic chromatography step, and a second anion chromatography step) and two diafiltrations (without additional purification or processing before distribution and storage). A 1000 L scale GMP batch (1205114) was produced using multiple rounds of bind-and-elute chromatography (including a first anion chromatography step, a hydrophobic chromatography step, and a second anion chromatography step) and two diafiltrations.

[0381] Briefly, both the 500 and 1000 L scale involve the following steps: a) Inoculum growth (sufficient viable cells serve as inoculum for the production bioreactor through serial subculture). The contents of each vial of the cell bank are thawed and the cells are sedimented by centrifugation. After resuspension in warmed growth medium, the cells are transferred to shake flasks. The culture is propagated by subculture in shake flasks and then roller bottles using growth medium in an atmosphere of 5% CO2 in air until sufficient cells are generated to inoculate the single-use inoculum bioreactor. The inoculum is further propagated in subsequent inoculum bioreactors until sufficient cells are generated to inoculate the single-use production bioreactor; b) Cell culture in a production bioreactor (either 500 L or 1000 L) (the production bioreactor process is to promote cell growth under controlled conditions to express recombinant protein of desired quality that can then be purified from the harvest filtrate during subsequent downstream processing steps). Prior to cell culture, the required amount of growth medium is added to the production bioreactor and equilibrated with respect to pH, temperature, and dissolved oxygen concentration, and then the cells are aseptically transferred from the inoculum bioreactor to the production bioreactor. Throughout the cell culture process, various additives and feeds are aseptically added to the bioreactor according to the feed addition standards; c) Harvesting (removing cells and cell debris and providing a suitable product stream for the purification process). The production bioreactor is cooled prior to harvesting, and then cells and cell debris are removed by filtering the culture through depth filters ranging in nominal pore size from 9.0 to <0.1 μm. The clarified supernatant is then aseptically filtered through a 0.22 μm filter into a sterile bioprocess vessel; d) TDAO treatment and anion exchange chromatography (Toyopearl GigaCap Q 650M) (N,N-dimethyltetradecylamine N-oxide (TDAO) is added to the clarified harvest). This is the first of two specific viral inactivation / reduction steps in the process. The first anion exchange chromatography serves as the main capture step in the downstream purification process. The principle of this step is anion exchange bind and elute chromatography, where the resin selectively binds to proteins, allowing impurities to flow through the column. The treated harvest supernatant is loaded onto an anion exchange column. After loading, the column is washed, allowing impurities to be removed, and then the bound proteins are eluted as a single fraction using a high-salt buffer; e) Hydrophobic Interaction Chromatography (HIC, Capto Phenyl, High Sub) (the resin binds the protein and allows impurities to flow through the column). The in-process product is diluted and loaded onto the HIC column. After loading, the column is washed to allow impurities to be removed, and then the bound protein is eluted as a single fraction by decreasing the conductivity of the buffer; f) Concentration and Diafiltration (The purpose of the step is to exchange the buffer of the in-process product and adjust the protein concentration in preparation for the next chromatography step). The in-process product is concentrated using an ultrafiltration unit containing a single-use 30 kDa molecular weight cut-off cassette. The concentrate is then diafiltered in preparation for the next process step; g) Anion exchange chromatography (Toyopearl GigaCap Q 650S) (the resin selectively binds proteins, allowing impurities to flow through the column). The concentrated and diafiltered product is loaded onto an anion exchange column. After loading, the column is washed, allowing impurities to be removed, and then the bound protein is eluted as a single fraction by increasing the buffer conductivity; h) Virus reduction filtration (in-process product is filtered through a small single-use virus-retaining filter (Planova BioEx) for the purpose of physically removing adventitious viruses, if present). Integrity testing is performed on the filters before and after use; i) Diafiltration into bulk formulation buffer (the purpose of the step is to buffer exchange the in-process product in preparation for the next processing step). The in-process product is processed using an ultrafiltration unit containing a single-use 30 kDa molecular weight cut-off cassette. The product is then diafiltered into bulk formulation buffer; j) excipient addition (excipient buffers are added to the diafiltered in-process product at specific ratios to obtain a final formulation of 20 mM Tris, 50 mM sodium chloride, 10 mM L-methionine, 150 mM sucrose, 0.02% (w / v) polysorbate 20, pH 7.3); and k) Bulk Filtration, Filling and Storage (Bulk drug substance is 0.22 μm filtered and aseptically filled into sterile containers (Pall Allegro) and stored). After use, filters are integrity tested. Drug substance is stored at -65°C or below.

[0382] A batch analysis summary for the IL-12 fusion polypeptide drug substance is shown in Table 42.

[0383] [Table 42-1]

[0384] [Table 42-2]

[0385] Features The liquid color, clarity and opacity, protein concentration, osmolality, and pH results for the IL-12 Fusion Polypeptide Analytical Reference Standard (500 L batch) and the IL-12 Fusion Polypeptide GMP (1000 L batch) batch met all specifications. The results for the IL-12 Fusion Polypeptide Analytical Reference Standard were comparable to those of the GMP drug substance batch. See Table 42.

[0386] Identity and Purity Reduced RP HPLC was performed on the IL-12 fusion polypeptide reference standard and the IL-12 fusion polypeptide GMP drug substance batch to assess pre-peak, main peak, and post-peak levels as part of batch release testing. The area percentages of the main peak, Group A, and B peaks for the IL-12 fusion polypeptide GMP drug substance batch and the IL-12 fusion polypeptide reference standard are shown in Table 43. Chromatograms for the GMP batch and reference standard are shown in Figure 18.

[0387] [Table 43]

[0388] The major peak was Peak 2 and was labeled as the major peak. A group of Peak A and a group of Peak B were detected for both samples. The RP HPLC profile of the GMP drug substance batch was comparable to the reference standard. All results met the current GMP specifications at the time of testing of ≥ 60.0% area of ​​the major peak and reported % of the areas of the Group A and B peaks. The identity of the GMP drug substance batch was also confirmed as comparable to the reference standard (Table 42).

[0389] activity The biological activity (interleukin bioassay) of the IL-12 fusion polypeptide drug substance for the GMP batch was 108% compared to the IL-12 fusion polypeptide reference standard. Results for the IL-12 fusion polypeptide reference standard (100% compared to the product standard) and the GMP drug substance batch (108% compared to the IL-12 fusion polypeptide reference standard) were comparable (Table 42).

[0390] purity Size Heterogeneity (Aggregation) - SE HPLC SE HPLC was performed on the IL-12 fusion polypeptide reference standard and GMP drug substance batches to assess the levels of main peak, high molecular weight species (HMW), and low molecular weight species (LMW) as part of batch release testing. The results for % main peak, HMW, and LMW are shown in Table 44, and the chromatograms are shown in Figure 20.

[0391] [Table 44]

[0392] One high molecular weight species was detected for both the IL-12 fusion polypeptide reference standard and the IL-12 fusion polypeptide GMP drug substance batch (HMW1). Slightly higher levels of HMW peaks were seen in the GMP drug substance batch compared to the reference standard. This was consistent with results from sedimentation velocity analytical ultracentrifugation (SV-AUC), where HMW species were more consistently detected at higher levels for the GMP drug substance batch. No LMW species were detected for both samples.

[0393] Results for the SE HPLC analysis demonstrated low aggregation and that the GMP drug substance batch was comparable to the reference standard (Table 42).

[0394] Aggregation measured by anion exchange (AEX) HPLC AEX HPLC was performed on the IL-12 fusion polypeptide reference standard and GMP drug substance batches to assess the levels of major peaks 1 and 2, acidic and basic peaks as part of the batch release characterization. The results are shown in Table 45 and the chromatograms are shown in Figure 21.

[0395] [Table 45]

[0396] Three peaks were detected in both samples. The total relative percentages of major peak 1, major peak 2, and the acidic peak were calculated. A slightly higher major peak 1 and a slightly lower major peak 2 were observed for the GMP drug substance batch when compared to the reference standard. However, the sum of the major peaks was comparable between samples. This may be related to the slightly lower level of phosphorylation determined for the GMP drug substance batch by malachite green phosphoprotein estimation. However, the sum of the major peaks was comparable between samples. For the AEX HPLC analysis, results demonstrated low aggregation and that the GMP drug substance batch was comparable to the reference standard by AEX HPLC.

[0397] Phosphoprotein Phosphate Estimation Assay (Malachite Green) The levels of phosphorylation for the IL-12 fusion polypeptide reference standard and the IL-12 fusion polypeptide GMP drug substance batch were 8.0 and 7.1, respectively, and the results were comparable. This is consistent with findings from reverse-phase high-performance liquid chromatography (RP HPLC) reduced tryptic peptide mapping and mass isoform heterogeneity by electrospray ionization mass spectrometry (ESI-MS). The manufactured IL-12 fusion polypeptides exhibited the appropriate degree of phosphorylation (Table 42).

[0398] Fragmentation measured by reduced capillary electrophoresis sodium dodecyl sulfate assay (CE-SDS) The electrophoretic patterns for the IL-12 fusion protein reference standard and the GMP drug substance batch were evaluated using capillary electrophoresis sodium dodecyl sulfate (CE-SDS) performed under reducing conditions as part of the batch release study. The purity percentages for the GMP drug substance batch and reference standard were 97.4% and 95.4%, respectively (Table 42). The chromatograms for the GMP batch and reference standard are shown in Figure 19.

[0399] The major peak for both samples met the GMP specification of ≥90.0%, which was current at the time of testing. One additional minor peak (peak 4) was detected in the reference standard, while one additional minor peak (peak 6) was detected in the GMP drug substance batch. However, the profiles were comparable, with variability for the minor peak below the limit of quantitation (LOQ). All results met the current GMP specification at the time of testing, and the data demonstrated that the IL-12 fusion polypeptide GMP drug substance batch was comparable to the reference standard by reduced CE SDS analysis. The IL-12 fusion polypeptide reference and drug substance exhibited low fragmentation and high purity (Table 42).

[0400] Impurities (process) The IL-12 fusion polypeptide reference standard and GMP drug substance batches were tested for Chinese hamster ovary (CHO) deoxyribonucleic acid (DNA) by quantitative polymerase chain reaction (qPCR) and CHO host cell protein (HCP) enzyme-linked immunosorbent assay (ELISA) as part of batch release testing. Levels of process-related impurities for the GMP drug substance batches were comparable to the reference standard (Table 42).

[0401] safety Bioburden and endotoxin testing for the IL-12 fusion polypeptide reference standard and GMP drug substance batches was performed as part of batch testing. Safety results for the GMP drug substance batches were comparable to the reference standard (Table 42).

[0402] Example 11: Characterization of exemplary IL-12 fusion polypeptide drug substances This example provides the characterization of an exemplary IL-12 fusion polypeptide drug substance. This example demonstrates IL-12 fusion polypeptides with a degree of phosphorylation of 7.1 or 8.

[0403] ESI-MS for mass spectrometry Relative molecular masses were determined via ESI-MS. IL-12 fusion polypeptide drug substances contain high heterogeneity in terms of product variants due to the presence of multiple different sialylated glycans (N-linked and O-linked glycans) and phosphorylation sites. Therefore, the mass of intact IL-12 fusion polypeptide drug substances could not be determined directly by ESI-MS. Instead, heterogeneity had to be reduced by removing at least the glycans (deglycosylation) with an enzyme mixture containing N-glycosidase (PNGaseF), sialidase (SialExo), and O-glycanase (OglyZOR). This was performed under reducing and denaturing conditions to increase the exposure of these post-translational modifications to the enzyme. Deglycosylated product samples were prepared with or without dephosphorylation with lambda phosphatase and analyzed by ESI-MS with online desalting. Using this approach, spectra were obtained that confirmed the major masses of the deglycosylated products with and without phosphorylation. To obtain additional information regarding the distribution of differentially phosphorylated / lower intensity species, samples of the deglycosylated product samples were also analyzed by a second ESI-MS method with coupled RP HPLC to provide the necessary resolution.

[0404] Results for ESI-MS with coupled desalting Deglycosylated IL-12 fusion polypeptide drug substance in-process (before excipients) samples were analyzed by ESI-MS with coupled desalting. The deconvoluted spectra are shown in Figure 22. The major masses detected for the deglycosylated IL-12 fusion polypeptide drug substance in-process samples for the reference batch and GMP batch were 64,151 Da and 64,152 Da (species 3), respectively, which was consistent with the expected theoretical mass (64,154 Da) of deglycosylated IL-12 fusion polypeptide drug substance with nine phosphorylations. The theoretical mass was the theoretical amino acid sequence for the IL-12 fusion polypeptide drug substance with fully reduced disulfide bonds and asparagine to aspartic acid conversion for all potential N-linked glycosylation sites as a result of enzymatic deglycosylation. Masses were assigned to a total of five species with masses consistent with varying degrees of phosphorylation (the theoretical mass difference (delta mass) for phosphorylation is +80 Da).

[0405] The ESI-MS spectra are visually comparable and confirm similar levels of phosphorylated species for the GMP drug substance batch and the reference standard batch (a representative in-process, pre-excipient sample), consistent with findings from malachite green phosphoprotein phosphate estimation and RP HPLC tryptic peptide mapping.

[0406] The deglycosylated and dephosphorylated IL-12 fusion polypeptide drug substance in-process samples were analyzed by ESI-MS with coupled desalting. Dephosphorylation was incomplete but sufficient to reveal species consistent with the underlying protein masses. The deconvoluted spectra are shown in Figure 22. The major masses detected for the deglycosylated and dephosphorylated IL-12 fusion polypeptide drug substance in-process samples for the reference batch and GMO batch were 63,513 Da and 63,514 Da (species 2), respectively, which was consistent with the expected theoretical mass of a deglycosylated IL-12 fusion polypeptide drug substance with one phosphorylation (63,514 Da). The masses of the deglycosylated and fully dephosphorylated IL-12 fusion polypeptide drug substance protein chains were detected at 63,442 Da and 63,441 Da (species 1), respectively, for both in-process samples for the reference batch and GMO batch (theoretical mass 63,434 Da). Species 3 was observed for both samples, which is consistent with a deglycosylated IL-12 fusion polypeptide drug substance with two phosphorylated or hexose sugars, which may indicate glycation (glycation was confirmed by RP HPLC reduced tryptic peptide mapping). ESI-MS spectra were visually comparable between the reference batch and GMO batch in-process samples.

[0407] Results for ESI-MS with coupled RP HPLC The deglycosylated IL-12 fusion polypeptide reference standard and GMP drug product batch were analyzed by ESI-MS with coupled RP HPLC. The total ion current chromatograms are shown in Figure 23, and the deconvoluted spectra are shown in Figure 24. The major masses detected for the reference standard and GMP drug product batch were 64437.6 Da and 64437.1 Da, respectively, consistent with the mass of the IL-12 fusion polypeptide drug substance (theoretical mass 64437.1 Da) with no N-linked glycans but with an O-linked glycan (HexHexNAc) and eight phosphorylations. This suggested that the O-glycanase treatment was incomplete in this assay, or that O-glycosylated species were better detected in this method by coupled RP HPLC, which reduces background matrix effects. Other masses were consistent with varying degrees of phosphorylation, with or without a single O-linked glycan. The occupancy and level of phosphorylation observed was comparable between the two batches and consistent with that determined by RP HPLC reduced tryptic peptide mapping and malachite green phosphoprotein phosphate estimation.

[0408] ESI-MS with coupled RP HPLC revealed a broader spectrum of product phosphorylations (5–12 phosphorylations; the major species had 8 phosphorylations) than that determined by ESI-MS with coupled desalting (7–11 phosphorylations; the major species had 9 phosphorylations). This is likely due to differences in species resolution due to the different mass spectrometers used for each method and differences in interference from the sample matrix (better removal of interfering / ion-suppressing entities by RP HPLC compared to desalting alone). Overall, both approaches confirmed that the masses of the batches were as expected and that the product manifold profiles were comparable between the two batches.

[0409] RP HPLC-MS and MS / MS reduced tryptic peptide mapping Test samples were denatured with guanidine hydrochloride and reduced with tris-(2-carboxyethyl)-phosphine (TCEP). The samples were then buffer-exchanged into a urea / TCEP buffer for trypsin digestion to maintain both the enzymatic activity and solubility of the molecules. The samples were then digested using trypsin and separated using RP HPLC. A combination of mass spectroscopy and ultraviolet (UV) detection (λ210 nm and λ280 nm) was used.

[0410] 210 nm, 280 nm and total ion count (TIC) profiles were generated. The TIC chromatograms for the IL-12 fusion polypeptide reference standard and the GMP drug substance batch were visually comparable.

[0411] Sequence coverage Signals from the acquired mass spectra matched the theoretical tryptic digestion profile for the IL-12 fusion polypeptide drug substance. 100% sequence coverage for the IL-12 fusion polypeptide reference standard and GMP drug substance batches was confirmed by MS and supported by MS / MS assignments in the form of intact and partially digested (miss-cleaved) peptides using a combination of untreated and dephosphorylated samples.

[0412] N-terminal sequence N-terminal sequence verification was performed on the IL-12 fusion polypeptide reference standard and GMP drug substance batches using mass spectrometry and tandem mass spectrometry detection. The expected N-terminal peptide sequence (IWELKK, P1-2) was identified and confirmed by MS / MS. No N-terminal modifications were detected.

[0413] C-terminal sequence C-terminal sequence verification was performed on the IL-12 fusion polypeptide reference standard and GMP drug substance batches using mass spectrometry and tandem mass spectrometry detection. The C-terminal peptide (VMSYLNASGGGGEGGGGSEEGGGGSEEGGGGSEEGGGGSEEGGGGSEEGGGGSEEGGGGSEEGGGGSEEGGGGSEEGGGGSEEGGGGS, P59) was detected in both samples under dephosphorylated conditions as an unmodified and monophosphorylated peptide.

[0414] Post-translational modifications For the IL-12 fusion polypeptide reference standard and GMP drug substance batches, estimates of the amount of potential post-translational modifications were assigned by comparing the ion intensities from the modified peptide with those from the unmodified peptide. The results must be considered approximate, as chromatographic and intensities of low abundance modified peptides also cannot be assumed to have the same ionization properties as the unmodified peptide.

[0415] deamidation Ions consistent with potential deamidated variants were observed for the IL-12 fusion polypeptide reference standard and GMP drug substance batches. Putative deamidated peptides have molecular masses 1 Da higher than the corresponding intact peptides due to the substitution of amides on asparagine (N) or glutamine (Q) residues with carboxyl groups (conversion to aspartic acid / isoaspartic acid and glutamic acid residues, respectively). Typically, peptides prone to deamidation have a high frequency of both N and Q residues flanked by serine (S) and glycine (G) residues, which have been suggested to stabilize intermediates in the deamidation reaction.

[0416] Ions consistent with potential deamidation were detected for P17 at 1% for both samples. The level of deamidation for the GMP drug substance batch was comparable to the reference standard.

[0417] oxidation Ions consistent with potential oxidation variants of the IL-12 fusion polypeptide reference standard and GMP drug substance batch were observed. The putative oxidized peptides had molecular masses of 16 Da higher than the corresponding intact peptides, suggesting oxidation of methionine (M) or tryptophan (W).

[0418] Three signals consistent with potential oxidation were detected for both samples. The level of oxidation detected for the GMP drug substance batch was comparable to the reference standard.

[0419] Glycosylation Assessment of potential glycosylation site occupancy and heterogeneity was performed using tryptic digests of the IL-12 fusion polypeptide reference standard and GMP drug substance batches. The theoretical amino acid sequence of the IL-12 fusion polypeptide drug substance displayed seven sequence motifs typically associated with N-linked glycosylation (Asn-X-Ser / Thr (X is any residue except proline)) at Asn103, Asn113, Asn200, Asn281, Asn392, Asn406, and Asn516. N-linked glycans were detected at five of the seven potential N-linked glycan sites: Asn103, Asn200, Asn281, Asn392, and Asn406. Glycosylation was not detected at the potential N-linked glycosylation sites Asn113 and Asn516. There was also evidence for O-linked glycan attachment. Although O-linked glycans are known to be typically attached through the side chains (hydroxyl groups) of serine and threonine residues, the exact site of O-linked glycosylation could not be predicted from the sequence. One peptide that contained O-linked glycosylation was identified from tryptic digest data. N-linked and O-linked glycosylation site occupancy for the GMP drug substance batch was comparable to the reference standard.

[0420] phosphorylation IL-12 fusion polypeptide drug substances are believed to be phosphorylated by co-expressed human kinase Fam20C. Phosphorylation by human kinase Fam20C was assessed for IL-12 fusion polypeptide drug substances by reduced tryptic peptide mapping RP HPLC-MS. The estimated level of phosphorylation at each site is given in Table 46.

[0421] [Table 46]

[0422] Phosphorylation sites were detected at Ser43, Ser154, Ser168, Ser281, Ser306, Ser311, Ser316, Ser406, and Ser481 for the GMP drug substance batch and reference standard. Phosphorylation was detected at 1% for the Ser365 reference standard but not for the GMP drug substance batch. For both batches, no phosphorylation was detected at potential phosphorylation sites Ser233, Ser491, Ser498, Ser505, Ser512, Ser520, Ser527, Ser534, and Ser541.

[0423] Overall, the levels of phosphorylation for the GMP drug substance batches were comparable to the reference standard, consistent with findings from malachite green phosphoprotein phosphate estimation by ESI-MS and mass isoform heterogeneity by ESI-MS.

[0424] Glycosylation More glycosylation occurs most readily at the N-terminal alpha-amino groups of proteins or at the epsilon-amino groups of lysine residues throughout the protein sequence.

[0425] Data searches were performed on the reduced tryptic peptide mapping MS and MS / MS data to determine whether there was evidence for hexose sugar attachment, which would indicate glycation. Given that trypsin would not cleave at glycated lysine residues, data searches were performed on tryptic peptides that had internal lysine residues (i.e., incompletely cleaved peptides) and a single hexose attached mass (162 Da). The level of glycation could not be determined from peptide mapping alone because it relied on partially digested peptides. Therefore, these data alone indicate that glycation was detected; the exact mass and level are not reported in this study.

[0426] Detection of glycation for GMP drug substance batches was comparable to the reference standard.

[0427] Neutral oligosaccharides Neutral and charged N-linked oligosaccharide profiles were determined using HPLC and LC-MS methods. N-linked oligosaccharides were released by incubation with the enzyme peptide-N-glycosidase F (PNGase F) under denaturing and reducing conditions. Protein was removed, and the released oligosaccharides were ready for neutral and charge profiling. Released glycans were labeled with the fluorophore 2-aminobenzamide (2-AB) and incubated with α-sialidase to remove terminal sialic acids. Neutral profile—N-linked oligosaccharides were released by incubation with recombinant peptide-N-glycosidase F (PNGase F) under native conditions. Protein was removed by precipitation, and the released oligosaccharides were hydrolyzed. Oligosaccharides were labeled with the fluorophore 2-aminobenzamide (2-AB) and treated with α-sialidase prior to analysis. Glycan profiles and peaks are quantified by fluorescence peak area and identified by LCMS.

[0428] The N-linked oligosaccharide profiles of the IL-12 fusion polypeptide reference standard and GMP drug substance batches were determined using LC-MS of fluorescently labeled glycans released using PNGase F. The chromatograms are shown in Figure 25.

[0429] Proposed oligosaccharide structures were assigned based on accurate mass measurements, with further confirmation by retention time alignment with N-glycan standards and, where appropriate, IgG IAC to aid identification.

[0430] The detected N-glycans were consistent with predominantly core-fucosylated, biantennary, complex-type glycan structures with varying levels of terminal galactosylation, as expected for the GS-CHO cell line. The predominant N-glycan detected was G2F (peak 14). Other major N-glycans detected were two isomers of G3F (peaks 23 and 24), Man-9 (peak 27), two isomers of G4F (peaks 29 and 30), Man-6 (peak 11), G0F (peak 4), and two isomers of G1F (peaks 8 and 9). The results from N-linked oligosaccharide profiling were consistent with those from peptide mapping.

[0431] Although some variation in the levels of minor glycans was observed, overall the N-linked oligosaccharide profile of the IL-12 fusion polypeptide GMP drug substance batch appeared comparable to the reference standard based on the absence of additional glycan structures compared to the current reference standard and similar levels of major glycans.

[0432] charged oligosaccharides Charge profile: 2-AB-labeled glycans were separated according to charge by HPLC. Sialylated glycans were identified by comparison with the retention times of mono-, di-, tri-, and tetra-sialylated external standards. The relative percentage of each charged group was calculated based on the area of ​​each peak. Sialidase treatment was also performed to confirm that the charged groups were due to the presence of sialic acid.

[0433] The charged N-linked oligosaccharide profiles of the IL-12 fusion polypeptide reference standard and GMP drug substance batches were analyzed by hydrophilic interaction liquid chromatography (HILIC) of fluorescently labeled N-linked glycans. The identities of the reported charged groups were determined by comparison with test samples treated with α-sialidase. Neutral, monosialylated, disialylated, and trisialylated glycans were detected for both the IL-12 fusion polypeptide GMP drug substance batch and the reference standard, and the charged oligosaccharide profiles were comparable.

[0434] Alhydrogel / IL-12 fusion polypeptide drug substance binding kinetics and strength assay The manufacturing process for the IL-12 fusion polypeptide drug substance underwent development and scale-up to produce a 500 L toxicology lot (P4130826, current reference standard) and a 1000 L GMP lot (1205114). Samples from each lot were characterized using Alhydrogel binding kinetics and strength assays. Based on these results, the IL-12 fusion polypeptide drug substance remained comparable throughout the development of the manufacturing process.

[0435] The purpose of this assay is to measure the kinetics and binding strength of an IL-12 fusion polypeptide drug substance (IL-12-ABP) to Alhydrogel (aluminum hydroxide particles; alum). The IL-12-ABP protein contains a C-terminal fusion to a phosphorylated alum-binding peptide (ABP) and is expected to form a stable complex with Alhydrogel via a ligand exchange reaction between phosphoserine in the peptide and surface hydroxyl groups on the Alhydrogel. To test the stability (strength) of this complex, the bound protein is exposed to phosphoric acid and human serum (simulating in vivo conditions) to elute the weakly bound protein from the aluminum hydroxide. IL-12-ABP is included in the assay in the absence of Alhydrogel to serve as an unbound protein stability control.

[0436] IL-12-ABP (IL-12 fusion polypeptide drug substance) is conjugated to Alhydrogel by mixing the two components in a non-phosphate-containing IL-12-ABP formulation buffer at final concentrations of 0.25 mg / mL protein and 2.5 mg / mL aluminum hydroxide (1:10 ratio) for variable time intervals of 5, 10, and 30 minutes. To test Alhydrogel binding kinetics, samples from different time intervals are centrifuged to sediment all aluminum-bound protein particles. Free (unbound) IL-12-ABP in the supernatant is quantified by an IL-12 p70-specific sandwich ELISA assay (ELISA MAX™ Deluxe Set Human IL-12 (p70); BioLegend) (Table 47).

[0437] [Table 47]

[0438] IL-12-ABP is captured by a mouse IgG1 (R&D Systems) monoclonal antibody. Next, a biotinylated mouse monoclonal anti-human IL-12(p70) detection antibody, followed by avidin-HRP reagent and then TMB substrate, is added, producing a blue color proportional to the concentration of IL-12(p70) present. Each plate contains an eight-point free IL-12-ABP standard curve (each point in duplicate), and alum-bound IL-12-ABP conditions are included and analyzed in triplicate. To test the binding strength of IL-12-ABP's interaction with alum, alum-bound protein samples from 5-, 10-, and 30-minute intervals are diluted in 1 mM phosphate and 40% human serum and incubated at 37°C for 24 hours. At 2 and 24 hours, samples are removed and centrifuged to pellet the Alhydrogel particles. Free (unbound) IL-12-ABP in the supernatant is quantified as described above.

[0439] [Table 48]

[0440] Example 12: Impurities - Permissible Intravenous Daily Exposure (PDE) IV ) This example demonstrates that unwanted process impurities (e.g., TDAO, tropolone, Pluronic, PDMS, octamethylcyclotetrasiloxane D4, and Fam20) are removed during the manufacturing process of an IL-12 fusion polypeptide drug substance. Low molecular weight molecules (e.g., octamethylcyclotetrasiloxane D4 and tropolone) are removed during multiple rounds of bind-and-elute chromatography and two diafiltrations. High molecular weight compounds (PDMS, Pluronic, TDAO) are expected to be removed from the process during three bind-and-elute chromatography unit operations. The manufacturing process is also capable of removing Fam20.

[0441] Process-related impurities, product-related impurities and contaminants that may be present in the IL-12 fusion polypeptide drug substance are measured in this example.

[0442] The following excipients are classified as process-related impurities identified as being of concern from a patient safety perspective that are not controlled during IL-12 fusion polypeptide drug substance manufacturing or as part of release testing. A toxicology evaluation was conducted to consider the risk posed to patients from exposure to these components.

[0443] Tropolone: ​​Tropolone is added to the production stage of the cell culture process as a nutrient feed. Tropolone is a lipid-soluble iron chelator that improves iron transport into cells, enabling cell growth and sustaining cell viability.

[0444] Pluronics: Pluronics are a component of the base powder used to prepare media for inoculum and production stage bioreactors. The purpose of this chemical is to improve cell membrane stability during cell culture.

[0445] Polydimethylsiloxane (PDMS) and Octamethylcyclotetrasiloxane D4: Polydimethylsiloxane (PDMS) and Octamethylcyclotetrasiloxane D4 are components of simethicone, the active ingredient in Antifoam C emulsion. Antifoam emulsion is typically added to cell cultures during production to control or inhibit foaming in the production bioreactor. Addition is variable and depends on the degree of foaming.

[0446] N,N-Dimethyltetradecylamine N-oxide (TDAO): N,N-Dimethyltetradecylamine N-oxide (TDAO) is a detergent added to clarified harvest supernatants for viral inactivation. This supplement is added to harvested cell culture fluids prior to the first chromatography step.

[0447] Fam20C: Human Fam20C is an endogenous Golgi-localized serine / threonine kinase that phosphorylates serine and threonine motifs on proteins. It is co-expressed with the ANK-101 drug substance to phosphorylate alum-binding peptides.

[0448] The Permitted Intravenous Daily Exposure (PDE) for the identified impurities tropolone, Pluronic, PDMS, octamethylcyclotetrasiloxane, D4, TDAO, and Fam20C IV ) limits were determined through toxicological evaluation and are summarized in Table 49.

[0449] [Table 49]

[0450] Removal Risk Assessment for TDAO, Tropolone, Pluronic, PDMS, and Octamethylcyclotetrasiloxane, D4 Defined PDE IVBased on this, a risk assessment calculation was performed to assess the residual risk of these potential impurities through downstream processing by determining how much of each impurity would be present in a dose of IL-12 fusion polypeptide drug substance if removal of each impurity was not required throughout the manufacturing process (maximum migration model). This assessment used a conservative approach based on the manufacturing process of the IL-12 fusion polypeptide drug substance batch.

[0451] The compound of interest is an IL-12 fusion polypeptide drug substance that binds to a PDE IV A certain number of log removals are required to ensure that the PDE is less than 90%. When possible, a 90% safety factor is used in removal, i.e., the amount of compound of interest remaining is less than the PDE. IV It is good practice to demonstrate that the level of potential impurities is not more than 10% of the PDE. IV The total number of log removals required to ensure that the log removal rate is below both the 90% safety factor and the 100% safety factor is summarized in Table 50.

[0452] [Table 50]

[0453] TDAO removal research Defined PDE IV Based on this, a risk assessment calculation was performed to determine how much TDAO would be present in a dose of IL-12 fusion polypeptide drug substance if removal of TDAO throughout the manufacturing process were not required. The assessment took a conservative approach based on the manufacturing process of an IL-12 fusion polypeptide drug substance batch and considered: (1) the amount of TDAO present in the culture (assuming no change in the total amount added to the bioreactor relative to the amount present after harvest), (2) the product titer at the date of harvest, (3) the maximum culture volume, (4) a worst-case cumulative downstream yield estimate of 14.2%, and (5) a clinical dose of 0.02 mg / kg (based on a maximum clinical dose of 1.25 mg IL-12 fusion polypeptide drug product and a 55 kg patient).

[0454] Based on this scenario, the amount of TDAO present in the 0.02 mg / kg dose is summarized in Table 51.

[0455] [Table 51]

[0456] The level of TDAO is PDE in IL-12 fusion polypeptide drug substance IV A certain number of log removals are required to ensure that the PDE IV is less than 10% of the PDE IV. When possible, it is good practice to demonstrate a 90% safety factor in removal, i.e., the amount of compound of interest remaining is 10% or less of the PDE IV. Furthermore, if the level of TDAO is below the PDE IV, IV The total number of log removals required to ensure that the log removal rate is below both the 90% safety factor and the 90% safety factor is summarized in Table 52.

[0457] [Table 52]

[0458] Determination of TDAO in in-process samples Quantification of TDAO was performed using reversed-phase liquid chromatography-mass spectrometry (RP-LC-MS / MS) detection, monitoring two MS / MS mass transitions. A calibration curve for the LC-MS / MS determination of the detergent was established by measuring at least five levels of calibration solutions. The regression function and correlation coefficient of the calibration curve were used to calculate the concentrations of the analytical samples.

[0459] To confirm the removal of the compound TDAO during downstream processing, intermediate samples from pilot and GMP batches were subjected to LC-MS / MS testing for TDAO. The buffer matrix for the samples was also analyzed. All samples were processed / analyzed in duplicate to confirm the content results for each sample (one of the duplicates was injected twice to confirm the reproducibility of the injection / analysis run).

[0460] All analyzed samples showed TDAO levels below the detection limit (limit of detection [LoD] = 0.3 mg / L). Capto Phenyl HS eluate samples were submitted for pilot and GMP batches, and results show removal of TDAO (below the detection limit) after two chromatographic steps and one intermediate ultrafiltration / diafiltration (UF / DF). Because TDAO levels were found to be below the detection limit, there is a high safety margin for TDAO removal for the current downstream process.

[0461] Conclusions of the impurity migration model for TDAO, tropolone, Pluronic, PDMS, and octamethylcyclotetrasiloxane, D4 It is expected that the required number of log removals will be achieved during the multiple rounds of bind-and-elute chromatography and two diafiltrations that comprise the process. Lower molecular weight compounds (octamethylcyclotetrasiloxane D4, tropolone) are expected to be removed from the process during the three bind-and-elute chromatography unit operations. Nearly all remaining compounds are expected to be removed in the permeate during the concentration and diafiltration stages of the intermediate UF / DF step.

[0462] Higher molecular weight compounds (PDMS, Pluronic, TDAO) are expected to be removed from the process during the three bind-and-elute chromatography unit operations. Any remaining compounds will behave in a similar manner to lower molecular weight compounds in the intermediate UF / DF as long as they are below their critical micelle concentration. In particular, TDAO levels were measured for different in-process samples after Capto Phenyl HS chromatography from three different batches and were found to be below the detection limit.

[0463] Quantification of Fam20 The amount of Fam20C was monitored by LC-MS / MS using a multiple standard addition approach. Fam20C was spiked at three levels in the analyte. The amount of Fam20C in each sample was measured using LC-MS / MS monitoring tryptic peptide 31. A linear regression of the measured levels in the spiked samples was then performed, and the original unknown amount of Fam20C was estimated by solving the equation of the fitted line at y=0. The Fam20C quantification results for the pilot and GMP batches are summarized in Table 53.

[0464] [Table 53]

[0465] The amount of Fam20C per mg of IL-12 fusion polypeptide drug substance in GigaCap Q-650M load is 0.01 mg / day, taking into account the maximum dose of IL-12 fusion polypeptide drug substance (1.25 mg / day). IV (1000 μg / d). At the end of the purification process, the amount of Fam20C per mg of IL-12 fusion polypeptide drug substance was reduced by a further two orders of magnitude, demonstrating that the purification process is capable of removing Fam20C.

[0466] equivalent Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the invention is not limited to the above description, but rather is set forth in the following claims.

Claims

1. 1. A method for producing a phosphorylated form of a fusion polypeptide, said fusion polypeptide comprising: (a) an immunomodulatory polypeptide comprising an immune agonist portion; and (b) a metal hydroxide-binding polypeptide the amino acid sequence of which contains multiple phosphorylation sites such that the fusion polypeptide can adopt phosphorylated and non-phosphorylated forms, The method includes co-expressing the fusion polypeptide in a host cell with a kinase that phosphorylates it, such that the fusion polypeptide and kinase are expressed at a ratio in the range of about 4:1 to about 10:

1.

2. The method of claim 1, wherein the fusion polypeptide comprises 6 to 10 phosphates.

3. The method of claim 1, wherein the fusion polypeptide comprises 7 to 9 phosphates.

4. 10. The method of claim 1, wherein the co-expressing step comprises expressing from a bicistronic construct.

5. 10. The method of claim 1, wherein the co-expressing step comprises expressing from a transposon-based plasmid.

6. The method of claim 5 , wherein the transposon-based plasmid is a DNA plasmid.

7. 6. The method of claim 5, wherein the cell is further transfected with a nucleotide sequence encoding a transposase enzyme.

8. 8. The method of claim 7, wherein the transposase enzyme is an integrase.

9. 9. The method of claim 8, wherein the transposase enzyme is a DDE / D integrase.

10. 8. The method of claim 7, wherein the nucleotide sequence encoding the transposase enzyme is RNA.

11. The method of claim 1 , wherein the nucleotide sequences encoding the fusion polypeptide and the kinase are integrated into the genome of the cell.

12. 10. The method of claim 9, wherein the integration results in a single copy integration per genomic locus.

13. 10. The method of claim 1, wherein the ratio is 8:

1.

14. 2. The method of claim 1, wherein the kinase is Fam20C kinase.

15. 15. The method of claim 14, wherein the Fam20C kinase has an amino acid sequence that is at least 80% identical to that of SEQ ID NO:

5.

16. The method of claim 1 , wherein the portion comprises an IL12 portion or a functional fragment thereof.

17. The method of claim 1 , wherein the immunoagonist portion comprises a first portion and a second portion or functional fragments thereof.

18. 18. The method of claim 17, wherein the first portion comprises an IL12 portion or a functional fragment thereof, and the second portion comprises an IL12 portion or a functional fragment thereof.

19. 19. The method of claim 18, wherein the first portion comprising the IL12 portion has an amino acid sequence that is at least 80% identical to that of SEQ ID NO:2, and the second portion comprising the IL12 portion comprises an amino acid sequence that is at least 80% identical to that of SEQ ID NO:

3.

20. 1. A method for producing a highly pure preparation of a phosphorylated form of a fusion polypeptide from a cell extract containing said phosphorylated form, comprising: The fusion polypeptide comprises: (a) an immunomodulatory polypeptide comprising an immune agonist portion; and (b) a metal hydroxide-binding polypeptide which amino acid sequence includes multiple phosphorylation sites such that it can adopt phosphorylated and unphosphorylated forms; further, the cell extract is from a cell expressing the fusion polypeptide and kinase in a ratio ranging from about 4:1 to about 10:1, such that the phosphorylated form is produced; The method does not include an affinity chromatography step.

21. The method comprises: an anion chromatography step; and Hydrophobic interaction chromatography step 21. The method of claim 20, comprising one or more purification steps selected from:

22. 1. A method for producing a highly pure preparation of a phosphorylated form of a fusion polypeptide from a cell extract containing said phosphorylated form, comprising: The fusion polypeptide comprises: (a) an immunomodulatory polypeptide comprising an immune agonist portion; and (b) a metal hydroxide-binding polypeptide, the amino acid sequence of which includes multiple phosphorylation sites such that it can adopt phosphorylated and unphosphorylated forms; and the cell extract is from a cell expressing the fusion polypeptide and kinase such that the fusion polypeptide and kinase are expressed in a ratio ranging from about 4:1 to about 10:1 such that the phosphorylated form is produced; The method comprises: an anion chromatography step; and Hydrophobic interaction chromatography step one or more purification steps selected from The method comprising:

23. 23. The method of claim 21 or 22, wherein the purification step comprises at least one anion chromatography capture step and at least one hydrophobic interaction chromatography step.

24. 23. The method of claim 21 or 22, wherein the purification step comprises at least two anion chromatography capture steps and at least one hydrophobic interaction chromatography step.

25. The purification step comprises: i) a first anion chromatography step; ii) a hydrophobic interaction chromatography step; and iii) Second Anion Chromatography Step 23. The method of claim 21 or 22, comprising:

26. 23. The method of claim 21 or 22, wherein the first anion chromatography capture step utilizes a low salt concentration to bind the phosphorylated fusion polypeptide to the chromatography column and a higher salt concentration to elute the phosphorylated fusion polypeptide.

27. 23. The method of claim 21 or 22, wherein the hydrophobic interaction chromatography step utilizes a high ammonium sulfate concentration to bind the phosphorylated fusion polypeptide to the chromatography column and a lower ammonium sulfate concentration to elute the phosphorylated fusion polypeptide.

28. 23. The method of claim 21 or 22, wherein the second anion chromatography step utilizes a low salt concentration to bind the phosphorylated fusion polypeptide to the chromatography column and a higher salt concentration to elute the phosphorylated fusion polypeptide.

29. 23. The method of claim 21 or 22, wherein the purification step comprises an initiation step of viral inactivation.

30. 30. The method of claim 29, wherein the viral inactivation step is carried out by contacting the fusion polypeptide with a detergent.

31. 26. The method of claim 25, wherein a first step of filtration is performed after the second hydrophobic interaction chromatography step.

32. 32. The method of claim 31, wherein the first step of filtration is an ultrafiltration / diafiltration step.

33. 26. The method of claim 25, wherein a second step of filtration is performed after the third anion chromatography capture step.

34. 34. The method of claim 33, wherein the second step of filtration is an ultrafiltration / diafiltration step.

35. i) a first nucleotide sequence encoding a fusion polypeptide, said fusion polypeptide comprising: a) an immunomodulatory polypeptide comprising an immunoagonist portion; and b) a metal hydroxide-binding polypeptide, the amino acid sequence of which includes multiple phosphorylation sites such that it can adopt phosphorylated and non-phosphorylated forms; the first nucleotide sequence comprising ii) a second nucleotide sequence encoding a kinase; 1. A mammalian cell engineered to contain the first nucleotide sequence and the second nucleotide sequence are in cis with each other and are each under the control of a regulatory nucleotide sequence that controls expression of the fusion polypeptide such that the expression of the fusion polypeptide is at a ratio of about 4:1 to about 10:1 with respect to expression of the kinase. The mammalian cell.

36. 36. The cell of claim 35, wherein the regulatory nucleotide sequence is a promoter.

37. 37. The cell of claim 36, wherein promoters of different strengths are used to achieve the desired ratio.

38. The cell of claim 37, wherein the fusion polypeptide is under the control of a CMV promoter or an EF1a promoter.

39. The cell of claim D4, wherein the kinase is under the control of an SV40 promoter or a Ubc promoter.

40. 36. A preparation of cultured mammalian cells according to claim 35.

41. 41. The preparation of claim 40, wherein the cells are monoclonal cells.

42. 41. The preparation of claim 40, wherein the cells are not monoclonal cells.

43. 43. The preparation of claim 42, wherein the expression of the fusion polypeptide and the kinase is reasonably equivalent to that of a monoclonal preparation of the same cell.

44. I) culturing engineered mammalian cells, said mammalian cells comprising: i) a first nucleotide sequence encoding a fusion polypeptide, said fusion polypeptide comprising: a) an immunomodulatory polypeptide comprising an immunoagonist portion; and b) a metal hydroxide-binding polypeptide, the amino acid sequence of which includes multiple phosphorylation sites such that it can adopt phosphorylated and non-phosphorylated forms; the first nucleotide sequence comprising ii) a second nucleotide sequence encoding a kinase; wherein the first nucleotide sequence and the second nucleotide sequence are in cis with each other and are each under the control of a regulatory nucleotide sequence that controls expression of the fusion polypeptide at a ratio of about 4:1 to about 10:1 with respect to expression of the kinase; II) purifying said phosphorylated fusion polypeptide without an affinity chromatography step; A method comprising:

45. The method comprises: an anion chromatography step; and Hydrophobic interaction chromatography step one or more purification steps selected from 45. The method of claim 44, comprising:

46. 1. A method for purifying a phosphorylated form of a fusion polypeptide from a cell extract containing said form, comprising the steps of: The fusion polypeptide comprises: (a) an immunomodulatory polypeptide comprising an immune agonist portion; and (b) a metal hydroxide-binding polypeptide, the amino acid sequence of which includes multiple phosphorylation sites such that it can adopt phosphorylated and unphosphorylated forms. The improvement includes: The above method, comprising performing the purification without an affinity chromatography step.

47. The improvement is 36. Performing said purification from a cell extract prepared from a culture of mammalian cells according to claim 35.

47. The improvement of claim 46, further comprising:

48. 1. A method for purifying a phosphorylated form of a fusion polypeptide from a cell extract containing said form, comprising the steps of: The fusion polypeptide comprises: (a) an immunomodulatory polypeptide comprising an immune agonist portion; and (b) a metal hydroxide-binding polypeptide, the amino acid sequence of which includes multiple phosphorylation sites such that it can adopt phosphorylated and unphosphorylated forms. The improvement includes: an anion chromatography step; and Hydrophobic interaction chromatography step carrying out said purification by a method comprising one or more steps selected from The method comprising:

Images