Method for preparing fusion polypeptide

Abstract

The present disclosure provides surprisingly useful methods of making fusion polypeptides, including immunomodulatory moieties and metal hydroxide binding moieties, as well as various related techniques, methods of making the fusion polypeptide metal hydroxide complexes and methods of using such fusion polypeptides and / or fusion polypeptide metal hydroxide complexes are included.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 424,047, filed on November 9, 2022, which is incorporated herein by reference in its entirety. Background Art

[0003] Useful fusion polypeptides have been developed in which a metal-binding polypeptide is conjugated to an immunomodulatory domain (e.g., an IL-12 immunomodulatory domain) (see, e.g., published international patent application WO 2020 / 263399). Specific uses of such fusion peptides include the treatment of certain medical conditions, such as cancer. Summary of the Invention

[0004] The present disclosure provides specific methods for making fusion polypeptides and formulations comprising such fusion polypeptides (specifically including, for example, IL-12 fusion polypeptides) (e.g., as described in published international patent application WO2020 / 263399). In some embodiments, the provided technology enables efficient and effective production of formulations of phosphorylated forms of such fusion polypeptides.

[0005] Among other things, the present disclosure identifies sources of problems with certain manufacturing technologies for preparing relevant fusion polypeptides (e.g., fusion polypeptides in which a metal-binding polypeptide is conjugated to an immunomodulatory domain (such as an IL-12 immunomodulatory domain), and in particular, phosphorylated forms of such fusion polypeptides). Without wishing to be bound by any particular theory, the present disclosure indicates that phosphorylated forms of fusion polypeptides may lead to and / or cause certain such manufacturing challenges. The present disclosure provides solutions to the problems identified, including particularly useful methods for making phosphorylated forms of fusion polypeptides. In some embodiments, such provided methods may include the step of co-expressing the fusion polypeptide with a kinase that phosphorylates the fusion polypeptide in a host cell, such that the fusion polypeptide and the kinase are expressed in a ratio ranging from about 4:1 to about 10:1. In some embodiments, the provided methods achieve the manufacture of a phosphorylated fusion polypeptide preparation characterized by a degree of phosphorylation (e.g., the average number of phosphate molecules per polypeptide) of about 7-9. Without wishing to be bound by any particular theory, the present disclosure indicates that too low phosphorylation can result in weaker binding to the metal hydroxide scaffold and may increase systemic leakage after administration of the cytokine:metal hydroxide complex to a patient; conversely, too high phosphorylation can result in reduced cytokine potency after binding to the metal hydroxide scaffold, for example, possibly due to structural changes. In some embodiments, the methods according to the present disclosure provide a phosphorylated form of a fusion polypeptide with enhanced metal hydroxide (e.g., alum) conjugation (e.g., enabling the production of its formulation).

[0006] 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., relative to methods using an affinity chromatography capture step and / or methods using host cells in which the fusion polypeptide and kinase are expressed at a ratio other than about 4:1 to about 10:1).

[0007] Among other things, the present disclosure demonstrates that typical methods for producing phosphorylated forms of fusion polypeptides via affinity chromatography do not achieve the extent of phosphorylation described herein. While affinity chromatography is generally expected in the art to be desirable, or even necessary, for the effective separation of phosphorylated forms of the relevant fusion polypeptides, the present disclosure surprisingly demonstrates that the provided methods lack any affinity chromatography step and can achieve superior results.

[0008] The present disclosure recognizes that methods including affinity chromatography steps can have various disadvantages, such as, for example, lengthy and / or expensive purification processes that often involve multiple steps that, together, can result in significant losses in product recovery. Furthermore, the present disclosure demonstrates that provided methods can achieve improved product quality (e.g., reduced levels of one or more undesirable substances, e.g., product-related substances) and / or reduced impurity load (e.g., host cell proteins, DNA) compared to certain conventional methods (including methods including one or more affinity chromatography steps).

[0009] The present disclosure provides particularly useful methods that achieve the production of high-purity preparations of phosphorylated fusion polypeptides as described herein. The provided technology provides improvements to various conventional manufacturing technologies and / or solves problems associated therewith. In certain embodiments, the provided manufacturing method (e.g., the specifically provided purification method) does not include an affinity chromatography capture step; alternatively or additionally, in some embodiments, the provided manufacturing method (e.g., the specifically provided purification method) includes one or more purification steps selected from an anion chromatography capture step; and a hydrophobic interaction chromatography polishing step. In some embodiments, the provided technology involves an anion chromatography step, followed by (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 technology is applied to cell extracts, for example, which can be prepared by engineered mammalian cells as provided in the present disclosure (e.g., engineered mammalian cells expressing a fusion polypeptide as described herein and a kinase that phosphorylates such a fusion polypeptide, such as engineered mammalian cells expressing a fusion polypeptide and a kinase in a range of about 4:1 to about 10:1, for example, at a ratio of about 8:1). BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1Exemplary schematic diagrams of the fusion polypeptide metal hydroxide complexes of the present disclosure are provided.The fusion polypeptide metal hydroxide complexes can be administered to a subject and result in enhanced retention and / or efficacy compared to an appropriate reference standard.

[0011] Figure 2 Schematic diagrams are provided of exemplary fusion polypeptides of the present disclosure comprising first (p40) and second (p35) IL12 immunoagonist portions and a metal hydroxide binding polypeptide having multiple phosphorylation sites. ABP: Alum Binding Peptide.

[0012] Figure 3A -C: shows the viability (A) and viable cell density (VCD) (B) of the stable pool during a 1 L fed-batch run. (C) shows the qualitative analysis of IL12 fusion protein by non-reducing sodium dodecyl polyacrylamide gel electrophoresis (NR SDS-PAGE).

[0013] Figure 4A -B: Shows viability (A) and viable cell density (B) during a 1 L fed-batch run of stable pools representing different IL12 fusion protein to FAM20C ratios. P1: Pool 6880D208_8:1; P2: Pool 6881D208_15:1; P3: Pool 6882D211_8:1; P4: Pool 6883D211_15:1; P5: Pool 6884D206_8:1; and P6: Pool 6885D209_8:1.

[0014] Figure 5 : Shows the productivity (titer) during a 1 L fed-batch run of stable pools representing different IL12 fusion protein to FAM20C ratios assessed by the biolayer interferometry (BLI) method.

[0015] Figure 6 : Shows the viability of the stable pool 6880P1 (8:1) after transfection and corresponding recovery.

[0016] Figure 7A -C: shows (A) viability, (B) viable cell density, and (C) titer during 1 L fed-batch culture of the stable pool 6880P1 (8:1).

[0017] Figure 8 : Shows the complete chromatogram and a zoom of the elution peaks during the first chromatography of the stabilized pool 6880P1 clarified harvest by anion exchange chromatography (GigaCap Q).

[0018] Figure 9 : Shows a qualitative analysis of anion exchange chromatography elution fractions on non-reducing SDS-PAGE. Fractions 13-25 were pooled. The volume was 18 mL and the concentration was 6.57 mg / mL.

[0019] Figure 10 : Shows the complete chromatogram and a zoom of the elution peaks during the first chromatography analysis of the stabilized pool 6880P1 clarified harvest by hydroxyapatite chromatography as a second chromatographic step.

[0020] Figure 11 : Shown is a qualitative analysis of hydroxyapatite chromatography fractions of 6880P1 on a non-reducing SDS-PAGE. 3 μg per lane. FT1 (lane 14) was acquired online midway through loading. FT2 (lane 15) was acquired online at the end of loading (maximum absorbance). Pool A: Fractions 6-9 from each run. 10 mL, concentration 2.63 mg / mL. Pool B: Fractions 10-13 from each run. 10 mL, concentration 1.30 mg / mL.

[0021] Figure 12 : shows the qualitative analysis of the hydrophobic interaction chromatography (phenyl) fraction of 6880P1 as the second chromatography step on non-reducing SDS-PAGE.

[0022] Figure 13A -C: shows the chromatogram (A) and qualitative analysis of the phenyl chromatography fractions of 6880P1 on non-reducing SDS-PAGE. The purity of the eluted fractions was determined by analytical size exclusion chromatography (SEC-HPLC) (B). The protein eluted with 0.5M AmSulfate is shown as a dotted line, or the protein eluted with 0.75M followed by 0.5M AmSulfate is shown as a solid line (C).

[0023] Figure 14 : Shows qualitative analysis of 6880P1 on non-reducing SDS-PAGE when both heparin affinity and hydrophobic interaction chromatography (phenyl) fractions were used as the second chromatography step. Non-reducing, no stain 4-20%, 3 μg per lane. Lane 2: Heparin 0.1 M E1, Lane 3: Heparin 0.1 M E2, Lane 4: Heparin 0.35 M E1, Lane 5: Heparin 0.35 M E2, Lane 6: Heparin 2 M E1, Lane 7: Phenyl 0.75 M E1, Lane 8: Phenyl 0 M E2, Lane 9: QHP loading.

[0024] Figure 15 : Shows qualitative analysis of Q HP anion exchange chromatography fractions of 6880P1 on non-reducing SDS-PAGE when used as the third chromatography step. Non-reducing, no stain, 4-20%, 3 μg per lane. Lane 2: Stripping, 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: Phenyl loading.

[0025] Figure 16A - B: shows the purity analysis of the heparin chromatography fraction (upper panel A), the phenyl chromatography fraction (lower panel A) and the Q HP anion exchange chromatography fraction of 6880P1 on SEC-HPLC when used as the first, second and third chromatography steps, respectively (B).

[0026] Figure 17 : Shows a chromatogram of ANK101 (IL-12 fusion polypeptide) purified after TOYOPEARL GigaCap Q-650S chromatogram during multiple cycles. The blue line is the A280nm trace of cycle 9, the orange line is the A280nm trace of cycle 10, the green line is the A280nm trace of cycle 11, and the purple line is the A280nm trace of cycle 12.

[0027] Figure 18 : Full view chromatogram showing reduced RP HPLC profile of IL-12 fusion polypeptide reference standard (top) and IL-12 fusion polypeptide GMP drug substance batch (bottom).

[0028] Figure 19 : Full view chromatogram showing the reduced CE-SDS electropherogram of an IL-12 fusion polypeptide reference standard (upper panel) and a GMP drug substance batch (lower panel).

[0029] Figure 20 : Full view showing SE HPLC chromatograms of IL-12 fusion polypeptide reference standard (upper panel) and GMP drug substance batch (lower panel).

[0030] Figure 21 : Full view showing AEX HPLC chromatograms of IL-12 fusion polypeptide reference standard (top) and GMP drug substance batch (bottom).

[0031] Figure 22 : Expanded view showing the deglycosylated ESI-MS processing spectrum of an IL-12 fusion polypeptide drug substance in-process sample before addition of excipient (polysorbate 20) for a reference standard (top) and a GMP drug substance batch (bottom).

[0032] Figure 23 : Shows total ion current chromatograms - deglycosylated sample of drug product GMP batch (top), reference standard (middle), and assay control (NEC) (bottom). The peak at RT≈16.5 min corresponds to a mass of approximately 34,778 Da and is assigned to PNGase F. Only the main peak was considered for data collection.

[0033] Figure 24: Shown are deconvoluted MS spectra of reduced and deglycosylated samples of a reference standard (lower panel) and a GMP batch of the drug product (upper panel).

[0034] Figure 25 : Full view showing the neutral N-linked oligosaccharide profiles of an IL-12 fusion polypeptide reference standard (top panel / P4130826ARS) and a GMP drug substance batch (bottom panel / 1205114).

[0035] definition

[0036] Administration: As used herein, the term "administration" generally refers to applying a composition to a subject or system. One of ordinary skill in the art will recognize various routes that can be used for administration to a subject (e.g., a human), where appropriate. For example, in some embodiments, administration can be systemic; in some embodiments, administration can be local. In some embodiments, administration can be enteral; in some embodiments, administration can be parenteral. In some embodiments, administration can be by injection (e.g., intramuscular, intratumoral, intravenous, or subcutaneous). In some embodiments, injection can involve a bolus, drip, perfusion, or infusion. In many embodiments, administration according to the present disclosure is performed by intratumoral injection.

[0037] Affinity: As known in the art, "affinity" is a measure of the tightness with which two or more binding partners associate with each other. Those skilled in the art are aware of a variety of assays that can be used to assess affinity, and are also aware of appropriate controls for such assays. In some embodiments, affinity is assessed in a quantitative assay. In some embodiments, affinity is assessed at multiple concentrations (e.g., concentrations of one binding partner at a time). In some embodiments, affinity is assessed in the presence of one or more potential competitor entities (e.g., which may be present in a relevant (e.g., physiological) environment). In some embodiments, affinity is assessed relative to a reference (e.g., a reference with a known affinity above a specific threshold [a "positive control" reference] or a reference with a known affinity below a specific threshold [a "negative control" reference]). In some embodiments, affinity can be assessed relative to a contemporaneous reference; in some embodiments, affinity can be assessed relative to a historical reference. Typically, when affinity is assessed relative to a reference, it is assessed under comparable conditions.

[0038] 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 phenomenon (e.g., heat, electric current or electric field, magnetic force or magnetic field, etc.). Where appropriate, as will be clear from the context to one skilled 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 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 man-made entities, in that it is designed, engineered, and / or produced by human action and / or is not found in nature. In some embodiments, the agent may be available in an isolated or pure form; in some embodiments, the agent may be available in a crude form. In some embodiments, potential agents may be provided as a collection or library, which, for example, may be screened to identify or characterize active agents therein.

[0039] Agonist: Those skilled in the art will appreciate that the term "agonist" may be used to refer to an agent, condition, or event whose presence, level, extent, type, or form is associated with an increase in the level or activity of another agent (i.e., an agonized or target agent). In general, an agonist may 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 the relevant activation activity. In some embodiments, an agonist may be direct (in which case it exerts its effect directly on its target, e.g., by physically binding to such a target); in some embodiments, an agonist may be indirect (in which case it exerts its effect by means other than binding to its target; e.g., by interacting with a modulator of the target, causing the level or activity of the target to be altered).

[0040] Amino Acid: In its broadest sense, as used herein, the term "amino acid" refers to a compound and / or substance that can be incorporated, is incorporated, or has been incorporated into a polypeptide chain, for example, by forming one or more peptide bonds. In some embodiments, an amino acid has the general structure H2N–C(H)(R)–COOH. In some embodiments, the amino acid is a naturally occurring amino acid. In some embodiments, the amino acid is a non-natural amino acid; in some embodiments, the amino acid is a D-amino acid; in some embodiments, the amino acid is an L-amino acid. "Standard amino acid" refers to any of the twenty standard L-amino acids commonly found in naturally occurring peptides. "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, the amino acids in a polypeptide (including amino acids at the carboxyl and / or amino termini) may contain structural modifications compared to the general structure described above. For example, in some embodiments, an amino acid may be modified by methylation, amidation, acetylation, pegylation, glycosylation, phosphorylation, and / or substitution (e.g., substitution of an amino group, a carboxylic acid group, one or more protons, and / or a hydroxyl group) compared to the general structure. In some embodiments, such modifications may, for example, alter the circulating half-life of a polypeptide comprising the modified amino acid, compared to a polypeptide comprising an otherwise identical unmodified amino acid. In some embodiments, such modifications do not significantly alter the relevant activity of a polypeptide comprising the modified amino acid, compared to a polypeptide comprising an otherwise identical unmodified 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, it may be used to refer to an amino acid residue of a polypeptide.

[0041] Animal: As used herein, the term "animal" refers to a member of the kingdom Animalia. In some embodiments, "animal" refers to a human of any sex and at any stage of development. In some embodiments, "animal" refers to a non-human animal of 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 worms. In some embodiments, an animal may be a transgenic animal, a genetically engineered animal, and / or a clone.

[0042] Binding: It will be understood that as used herein, the term "binding" generally refers to a non-covalent association between or among two or more entities. "Direct" binding involves physical contact between entities or moieties; indirect binding involves physical interaction by way of physical contact with one or more intermediate entities. Binding between two or more entities can generally be assessed in any of a variety of contexts, including studying the interacting entities or moieties alone or in the context of a more complex system (e.g., when covalently associated, electrostatically associated, 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 entities of interest are more likely to associate with each other than with other available binding partners.

[0043] Buffer: As used herein, the term "buffer" generally describes a solution whose pH varies based on its acid or base content. Buffer solutions resist changes in pH through the presence of their acid and base components. Typical buffers utilized in biological manufacturing processes maintain the pH of solutions within the physiological range. Conventional 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.

[0044] Cancer: The terms "cancer," "malignancy," "neoplasm," "tumor," and "cancer" are used herein to refer to cells that exhibit relatively abnormal, uncontrolled, and / or autonomous growth such that they exhibit an abnormal growth phenotype characterized by a significant loss of control over cell proliferation. In some embodiments, a tumor can be or comprise precancerous (e.g., benign), malignant, pre-metastatic, metastatic, and / or non-metastatic cells. The present disclosure specifically identifies certain cancers that may be particularly relevant to its teachings. In some embodiments, the relevant cancers may be characterized as solid tumors. In some embodiments, the relevant cancers may be characterized as hematologic tumors. In general, examples of different types of cancer known in the art include, for example, hematopoietic cancers, including leukemias, lymphomas (Hodgkin's lymphoma and non-Hodgkin's lymphoma), myelomas and myeloproliferative disorders; sarcomas, melanomas, adenomas, solid tissue cancers, squamous cell carcinoma of the mouth, pharynx, 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, thyroid cancer, parathyroid cancer, head and neck cancer, breast cancer, gastrointestinal cancer, and cancers of the nervous system, benign lesions such as papillomas, etc.

[0045] 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 the polymer. In some embodiments, the presence of a characteristic sequence element is associated with the presence or level of a specific activity or characteristic of the polymer. In some embodiments, the presence (or absence) of a characteristic sequence element defines a specific polymer as a member (or not a member) of a specific 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., continuously connected monomers). In some embodiments, a characteristic sequence element comprises at least a first and a second segment of continuous monomers, which are separated by one or more spacer regions, and for polymers sharing sequence elements, the length may or may not vary.

[0046] Chemotherapeutic agent: As used herein, the term "chemotherapeutic agent" has its art-understood meaning and refers to one or more pro-apoptotic, cytostatic, and / or cytotoxic agents, specifically including, for example, agents used and / or recommended for use in treating one or more diseases, conditions, or disorders associated with undesirable cell proliferation. In many embodiments, chemotherapeutic agents can be used to treat cancer. In some embodiments, the chemotherapeutic agent can 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, maytansines, and analogs thereof), one or more epothilones, one or more histone deacetylase inhibitors (HDACs), one or more topoisomerase inhibitors (e.g., topoisomerase I and / or topoisomerase II inhibitors), 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., which share relevant antiproliferative activity).In some specific embodiments, the chemotherapeutic agent can be or include one or more of the following: 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,

[00135] The present invention also includes but is not limited to dapoxetine, tadalafil, tadalafil, squalane, dapoxetine, squalane ... In some embodiments, chemotherapeutic agents may be used in the context of antibody-drug conjugates.In some embodiments, the chemotherapeutic agent is a chemotherapeutic agent found in an antibody-drug conjugate selected from the group consisting of 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, P4 / D10-doxorubicin, gemtuzumab ozogamicin, brentuximab vedotin, trastuzumab emtansine, and trastuzumab selenomethionine. 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, vorsetuzumab mafodotin, and lorvotuzumab mertansine. In some embodiments, the chemotherapeutic agent may be a chemotherapeutic agent as used in antibody-drug conjugates, such as described or discussed in one or more of Govindan et al., The Scientific World JOURNAL 10:2070, 2010,–2089). In some embodiments, the chemotherapeutic agent can be or comprise one or more of farnesyl-thiosalicylicacid (FTS), 4-(4-chloro-2-methylphenoxy)-N-hydroxybutyramide (CMH), estradiol (E2), tetramethoxystilbene (TMS), delta-tocotrienol, salinomycin, or curcumin.

[0047] Combination therapy: As used herein, the term "combination therapy" refers to those situations in which a subject is exposed to two or more treatment regimens (e.g., two or more therapeutic agents) simultaneously. In some embodiments, two or more regimens can be administered simultaneously; in some embodiments, such regimens can be administered sequentially (e.g., all "dosages" of the first regimen are administered before any doses of the second regimen are administered); in some embodiments, such agents are administered with overlapping dosing regimens. In some embodiments, "administration" of a combination therapy may involve administering one or more agents or modes to a subject who is receiving another one or more agents or modes in the combination. For clarity, combination therapy does not require that the individual agents be administered together in a single composition (or even necessarily administered simultaneously), but in some embodiments, two or more agents or their active portions 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).

[0048] Dosing regimen: Those skilled in the art will appreciate that the term "dosing regimen" can be used to refer to a collection of unit doses (usually more than one) that are individually administered to a subject, typically separated by time periods. In some embodiments, a given therapeutic agent has a recommended dosing regimen that may involve one or more doses. In some embodiments, the dosing regimen includes multiple doses, each dose separated in time from the other doses. In some embodiments, individual doses are separated by time periods of the same length; in some embodiments, the dosing regimen includes multiple doses and at least two different time periods separating the individual doses. In some embodiments, all doses within a dosing regimen have the same unit dose amount. In some embodiments, different doses within a dosing regimen have different amounts. In some embodiments, the dosing regimen includes a first dose of a first dose amount, followed by one or more additional doses of a second dose amount that is different from the first dose amount. In some embodiments, the dosing regimen includes a first administration of a first dose amount, followed by one or more additional administrations of a second dose amount that is the same as the first dose amount. In some embodiments, the dosing regimen is associated with a desired or beneficial outcome when administered to a relevant population (i.e., is a therapeutic dosing regimen).

[0049] Epitope: As used herein, the term "epitope" refers to a portion specifically recognized by an immunoglobulin (e.g., an antibody or a receptor) binding component. In some embodiments, an epitope is composed of multiple chemical atoms or groups on an antigen. In some embodiments, when the antigen adopts a relevant three-dimensional conformation, such chemical atoms or groups are surface exposed. In some embodiments, when the antigen adopts such a conformation, such chemical atoms or groups are physically close to each other in space. In some embodiments, when the antigen adopts an alternative conformation (e.g., linearization), at least some of such chemical atoms or groups are physically separated from each other.

[0050] 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, skim milk powder, glycerol, propylene, ethylene glycol, water, ethanol, and the like.

[0051] 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 may be a transcript. In some embodiments, the gene product may be a polypeptide. In some embodiments, expression of a nucleic acid sequence involves one or more of the following: (1) production of an RNA template from the 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.

[0052] Functional: As used herein, the term "functional" is used to refer to a form or fragment of an entity that exhibits a specific property and / or activity.

[0053] Fragment: A "fragment" of a material or entity as described herein has discrete portions comprising a whole, but lacks the structure of one or more portions found in the whole. In some embodiments, a fragment consists of such discrete portions. In some embodiments, a fragment consists of or contains characteristic structural elements or portions found in the whole. In some embodiments, a polymer fragment comprises 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, 100, 100, 100, 100, 100, 100, 100, 100, 100, 100, 100, 100, 100, 100, 100, 100, 100, 100, 100, 100, 100, 100, 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). In some embodiments, a polymer 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 entire polymer. In some embodiments, the entire material or entity can be referred to as the "parent" of the fragment.

[0054] 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 a coding sequence (i.e., a sequence that encodes a specific product); in some embodiments, a gene includes a non-coding sequence. In some specific embodiments, a gene may include coding (e.g., exon) sequences and non-coding (e.g., intron) sequences. In some embodiments, a gene may include one or more regulatory elements that, for example, may control or influence one or more aspects of gene expression (e.g., cell type-specific expression, inducible expression, etc.).

[0055] Gene product or expression product: As used herein, the term "gene product" or "expression product" generally refers to RNA transcribed from a gene (before and / or after processing) or a polypeptide encoded by RNA transcribed from a gene (before and / or after modification).

[0056] Genome: As used herein, the term "genome" refers to the total genetic information carried by a single organism or cell, represented by the complete DNA sequence of its chromosomes.

[0057] High molecular weight species (HMWS): as used herein, refers to dimers or multimers of a polypeptide (eg, fusion polypeptides). Such species can be identified, for example, by size exclusion chromatography (SEC)-HPLC.

[0058] Host cell: as used herein, refers to a cell into which exogenous DNA has been introduced (recombinantly or otherwise). A skilled artisan, after reading this disclosure, will understand that such terms refer not only to a particular subject cell, but also to the progeny of such a cell. Certain modifications may occur in subsequent generations due to mutations or environmental influences, and therefore such progeny may not actually 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 kingdom of life suitable for expressing exogenous DNA (e.g., a recombinant nucleic acid sequence). Exemplary cells include prokaryotic and eukaryotic cells (single or multicellular), bacterial cells (e.g., strains of Escherichia coli, Bacillus species, Streptomyces species, etc.), mycobacterial cells, fungal cells, yeast cells (e.g., Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris, Pichia 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 quadruple hybridomas). In some embodiments, the cell is a human, monkey, ape, hamster, rat, or mouse cell. In some embodiments, the cell is a eukaryotic cell and is selected from the group consisting of CHO (e.g., CHO K1, DXB-1 1CHO, Veggie-CHO), COS (e.g., COS-7), retinal cells, Vero, CV1, kidney (e.g., HEK293, 293EBNA, 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, MMT060562, Sertoli cells, BRL 3A cells, HT1080 cells, myeloma cells, tumor cells, and cell lines derived from the above cells. In some embodiments, the cell comprises one or more viral genes.

[0059] "Host cell protein" or "HCP": 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 in or on a host cell that produces a fusion polypeptide described herein (e.g., a phosphorylated or non-phosphorylated fusion polypeptide), and are not a fusion polypeptide. In some embodiments, the provided techniques (e.g., the provided manufacturing methods, such as the provided purification methods) exclude or reduce HCPs from preparations of fusion polypeptides (e.g., from preparations of phosphorylated fusion polypeptides as described herein). A "reduced HCP preparation" describes a preparation that contains reduced HCPs relative to, for example, the amount present before application of the relevant purification step (e.g., as provided herein) and / or relative to the amount achieved by a different purification technique. In some embodiments, the provided techniques enable the production of fusion polypeptide preparations (e.g., phosphorylated fusion polypeptide preparations) in which HCPs are undetectable, e.g., using, for example, an ELISA method. In some embodiments, the removal of HCPs can be monitored or assessed, for example, during or after purification of a fusion polypeptide as described herein (e.g., a phosphorylated form thereof), for example, from a host cell, which in some embodiments can be an engineered mammalian cell as described herein (e.g., which expresses the fusion polypeptide and the kinase that phosphorylates the fusion polypeptide in a ratio ranging from about 4:1 to 10:1, for example, at a ratio of about 8:1).

[0060] "Improve", "increase" or "decrease": as used herein, these terms or grammatically comparable comparative terms indicate values measured relative to a comparable reference. For example, in some embodiments, the evaluation value obtained with an agent of interest can be "improved" relative to the evaluation value obtained with a comparable reference agent. Alternatively or additionally, in some embodiments, the evaluation value obtained in a subject or system of interest can be "improved" relative to the evaluation value obtained under different conditions in the same subject or system (e.g., before or after an event such as administration of an agent of interest) or in different comparable subjects (e.g., in a comparable subject or system different from the subject or system of interest in the presence of one or more indicators of a particular disease, disorder or condition of illness, or in prior exposure to a condition of illness or agent, etc.). In some embodiments, comparative terms refer to statistically relevant differences (e.g., prevalence and / or magnitude sufficient to achieve statistical correlation). In a given context, one skilled in the art will recognize or will be able to readily determine the degree and / or prevalence of differences required or sufficient for achieving such statistical significance.

[0061] In vitro: As used herein, the term "in vitro" refers to events that occur in an artificial environment (eg, in a test tube or reaction vessel, in cell culture, etc.) rather than in a multicellular organism.

[0062] In vivo: as used herein refers to events that occur within a multicellular organism, such as humans and non-human animals. In the context of cell-based systems, the term can be used to refer to events that occur within living cells (as opposed to, for example, in vitro systems).

[0063] Isolated: as used herein, refers to a substance and / or entity that has been (1) separated from at least some of the components with which it was initially associated when it was produced (whether in nature and / or in an experimental setting) and / or (2) engineered, produced, prepared, and / or manufactured by the hand of man. An isolated substance and / or entity 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 it was initially associated. In some embodiments, the 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 more 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 still be considered "isolated" or even "pure" after having been 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 separation or purity percentage of a substance is calculated without including such carriers or excipients. In some embodiments, to give just one example, a biopolymer such as a polypeptide or polynucleotide occurring in nature is considered "isolated" when: a) by virtue of its origin or source of derivation, it is not associated with some or all of the components with which it accompanies it in its native state in nature; b) it is substantially free of other polypeptides or nucleic acids of the same species as the species in which it is produced in nature; or c) it is expressed by or otherwise associated with components from a cell or other expression system that does not have the species in which it is produced in nature. Thus, for example, in some embodiments, a polypeptide that is chemically synthesized or synthesized in a cellular system different from the cellular system in which it is produced in nature is considered an "isolated" polypeptide. Alternatively or additionally, in some embodiments, a polypeptide that has been subjected to one or more purification techniques may be considered an "isolated" polypeptide to the extent that it has been separated from other components a) with which it is associated in nature; and / or b) with which it is initially produced.

[0064] Linker: as used herein, is used to refer to the part that different elements are connected to each other in a multi-element agent. For example, it will be understood by those of ordinary skill in the art that a polypeptide whose structure includes two or more functional or organizational parts or domains typically includes a stretch of amino acids connecting them to each other between such parts or domains. In some embodiments, the polypeptide comprising a linker element has the overall structure of the general formula S1-L-S2, wherein S1 and S2 can be the same or different and represent two parts or domains that are associated with each other by a linker. In some embodiments, the length of the peptide 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 some embodiments, the joint is characterized in that it tends not to adopt a rigid three-dimensional structure, but rather provides flexibility to the polypeptide. Various different joint elements that can be appropriately used when engineering a polypeptide (e.g., a fusion polypeptide) 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).

[0065] Modulator: The term "modulator" is used to refer to an entity whose presence or level in a system where an activity of interest is observed is associated with a change in the level and / or nature of the activity compared to the activity observed under otherwise comparable conditions in the absence of the modulator. In some embodiments, the modulator is an activator, i.e., the activity is increased in the presence of the modulator compared to the activity observed under otherwise comparable conditions in the absence of the modulator. In some embodiments, the modulator is an antagonist or inhibitor, i.e., the activity is reduced in the presence of the modulator compared to otherwise comparable conditions in the absence of the modulator. In some embodiments, the modulator interacts directly with the target entity of the activity of interest. In some embodiments, the modulator interacts indirectly with the target entity of the activity of interest (i.e., directly with an intermediary that interacts with the target entity). In some embodiments, the modulator affects the level of the target entity of interest; alternatively or additionally, in some embodiments, the modulator affects the activity of the target entity of interest without affecting the level of the target entity. In some embodiments, the modulator affects both the level and activity of the target entity of interest such that the observed difference in activity is not fully explained by or is disproportionate to the observed difference in level.

[0066] Moiety: Those skilled in the art will understand that a "moiety" is a defined chemical group or entity having a specific structure and / or activity as described herein. Generally, a "moiety" is a portion of a molecule or entity that is smaller than its entirety.

[0067] Mutant: As used herein, the term "mutant" refers to an entity that exhibits significant structural identity to a reference entity but differs structurally from the reference entity in the presence or level of one or more chemical moieties compared to the reference entity. In many embodiments, a mutant is also functionally different from its reference entity. Generally speaking, whether a particular entity is properly considered a "mutant" of a reference entity is based on the degree of its structural identity to the reference entity. As will be appreciated by those skilled in the art, any biological or chemical reference entity has certain characteristic structural elements. By definition, a mutant is a unique chemical entity that shares one or more such characteristic structural elements. To name just a few examples, a small molecule can have a characteristic core structural element (e.g., a macrocyclic core) and / or one or more characteristic side group moieties such that a mutant of the small molecule is a small molecule that shares the core structural element and the characteristic side group moieties but differs in the type of bonds (single vs. double bonds, E vs. Z, etc.) present in the other side group moieties and / or within the core, a polypeptide can have a characteristic sequence element composed of a plurality of amino acids having designated positions relative to each other in linear or three-dimensional space and / or contributing to a specific biological function, a nucleic acid can have a characteristic sequence element composed of a plurality of nucleotide residues having designated positions relative to each other in linear or three-dimensional space. For example, a mutant polypeptide can differ from a reference polypeptide due to one or more differences in the amino acid sequence and / or one or more differences in the chemical moieties (e.g., carbohydrates, lipids, etc.) covalently attached to the polypeptide backbone. In some embodiments, the mutant polypeptide demonstrates an overall sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 99% with a reference polypeptide. Alternatively or additionally, in some embodiments, the mutant polypeptide does not share at least one characteristic sequence element with a reference polypeptide. In some embodiments, the reference polypeptide has one or more biological activities. In some embodiments, the mutant polypeptide shares one or more of the biological activities of the reference polypeptide. In some embodiments, the mutant polypeptide lacks one or more of the biological activities of the reference polypeptide. In some embodiments, the mutant polypeptide demonstrates a reduction in one or more biological activity levels compared to a reference polypeptide.

[0068] Operably linked: as used herein, refers to a juxtaposition wherein the components are in a relationship that permits them to function in their intended manner. A control element that is "operably linked" to a functional element is associated in such a manner 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 to (e.g., covalently linked to) a coding element of interest; in some embodiments, the control element acts in trans or otherwise on the functional element of interest.

[0069] Patient: As used herein, the term "patient" refers to any organism to which a provided composition is or can be administered, e.g., for experimental, diagnostic, preventive, 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 a human. In some embodiments, the patient suffers from or is susceptible to one or more conditions or illnesses. In some embodiments, the patient displays one or more symptoms of a condition or illness. In some embodiments, the patient has been diagnosed with one or more conditions or illnesses. In some embodiments, the condition or illness is or includes cancer, or the presence of one or more tumors. In some embodiments, the patient is receiving or has received certain therapies to diagnose and / or treat the disease, condition, or illness.

[0070] Pharmaceutical composition: As used herein, the term "pharmaceutical composition" refers to an active agent formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in a unit dosage amount suitable for administration in a treatment regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a population of interest. In some embodiments, the pharmaceutical composition can be specially formulated for administration in solid or liquid form, including pharmaceutical compositions suitable for a particular route of administration, for example, as described herein.

[0071] 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 excessive toxicity, irritation, allergic response, or other problem or complication, and are commensurate with a reasonable benefit / risk ratio.

[0072] 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 solvent encapsulating material, that participates in carrying or transporting the subject compound from one organ or part of the body to another organ or part of the body. 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 that can be used 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; powdered gum tragacanth; malt; gelatin; talc; excipients such as 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 glycerol, 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; ethanol; pH buffered solutions; polyesters, polycarbonates, and / or polyanhydrides; and other nontoxic, compatible substances employed in pharmaceutical formulations.

[0073] Polypeptide: As used herein, refers to a polymeric chain of amino acids. In some embodiments, a polypeptide has an amino acid sequence that occurs in nature. In some embodiments, a polypeptide has an amino acid sequence that does not occur in nature. In some embodiments, a polypeptide has an engineered amino acid sequence, meaning it is designed and / or produced through artificial action. In some embodiments, a polypeptide may comprise or consist of natural amino acids, non-natural amino acids, or both. In some embodiments, a polypeptide may comprise only natural amino acids, only non-natural amino acids, or only these amino acids. In some embodiments, a polypeptide may comprise D-amino acids, L-amino acids, or both. In some embodiments, a polypeptide may comprise only D-amino acids. In some embodiments, a polypeptide may comprise only L-amino acids. In some embodiments, a polypeptide may comprise one or more side groups or other modifications, such as at the N-terminus of the polypeptide, at the C-terminus of the polypeptide, or attached to one or more amino acid side chains, or any combination thereof. In some embodiments, such side groups or modifications may be selected from the group consisting of acetylation, amidation, lipidation, methylation, PEGylation, and the like, including combinations thereof. In some embodiments, a polypeptide may be cyclic and / or may contain a cyclic portion. In some embodiments, the polypeptide is not cyclic and / or does not contain any cyclic portions. In some embodiments, the polypeptide is linear. In some embodiments, the polypeptide can be or include a stapled polypeptide. In some embodiments, the term "polypeptide" can be appended to the name of a reference polypeptide, activity, or structure; in such cases, it is used herein to refer to polypeptides that share the relevant activity or structure and, therefore, can be considered members of the same class or family of polypeptides. For each such class, exemplary polypeptides within that class are provided herein and / or will be known to those skilled in the art, whose amino acid sequences and / or functions are known; in some embodiments, such exemplary polypeptides are reference polypeptides of that class or family of polypeptides. In some embodiments, members of a class or family of polypeptides exhibit significant sequence homology or identity with a reference polypeptide of that class (in some embodiments, with all polypeptides within that class), share common sequence motifs (e.g., characteristic sequence elements), and / or share common activities (in some embodiments, at comparable levels or within a specified range) with a reference polypeptide of that class (in some embodiments, with all polypeptides within that class).For example, in some embodiments, the member polypeptides exhibit an overall sequence homology or identity of at least about 30-40% and typically greater than about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more to the reference polypeptide, and / or include at least one region (e.g., a conserved region that may be or include characteristic sequence elements in some embodiments) that exhibits very high sequence identity (typically greater than 90% or even 95%, 96%, 97%, 98% or 99%). Such conserved regions typically encompass at least 3-4 and typically up to 20 or more amino acids; in some embodiments, conserved regions encompass at least a stretch of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more consecutive amino acids. In some embodiments, the polypeptide of interest may comprise or consist of fragments 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 spatial arrangement relative to each other that is different from that found in the polypeptide of interest (e.g., fragments that are directly linked in the parent may be spatially separated in the polypeptide of interest, and vice versa, and / or the fragments may be present in the polypeptide of interest in a different order than in the parent), such that the polypeptide of interest is a derivative of its parent polypeptide.

[0074] Predestined: Predestined means chosen intentionally, as opposed to occurring or happening randomly.

[0075] Pure: As used herein, an agent or entity is "pure" if it is substantially free of other components. For example, a formulation containing more than about 90% of a particular agent or entity is generally considered to be pure. 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.

[0076] Recombinant: As used herein, it is intended to refer to polypeptides designed, engineered, prepared, expressed, produced, manufactured and / or isolated by recombinant means, such as polypeptides expressed using recombinant expression vectors transfected into host cells; polypeptides isolated from a recombinant combinatorial human polypeptide library; polypeptides isolated from animals (e.g., mice, rabbits, sheep, fish, etc.) that are genetically modified or otherwise manipulated to express one or more genes or gene components that encode and / or direct the expression of a polypeptide or one or more components, parts, elements or domains thereof; and / or polypeptides prepared, expressed, produced or isolated by any other means involving splicing selected nucleic acid sequence elements or connecting selected nucleic acid sequence elements to each other, chemically synthesizing selected sequence elements and / or otherwise producing nucleic acids that encode and / or direct the expression of a polypeptide or one or more components, parts, elements or domains 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 by computer simulation. In some embodiments, one or more such selected sequence elements are produced by mutagenesis (e.g., in vivo or in vitro) of known sequence elements, e.g., from natural or synthetic sources such as, for example, in the germline of a source organism of interest (e.g., human, mouse, etc.).

[0077] Reference Standard: As used herein, describes a standard or control relative to which 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 at the same time as 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 will be understood by those skilled in the art, a reference or control is determined or characterized under conditions or circumstances comparable to those under evaluation. Those skilled in the art will understand when there is sufficient similarity to justify reliance on and / or comparison of a particular possible reference or control.

[0078] Specific binding: As used herein, the term "specific binding" refers to the ability to discriminate between possible binding partners in the context of binding. A binding agent that interacts with a particular target in the presence of other potential targets is said to "specifically bind" to the target with which it interacts. In some embodiments, specific binding is assessed by detecting or measuring the extent of association between the binding agent and its partner; in some embodiments, specific binding is assessed by detecting or measuring the extent of dissociation of the binding agent-partner complex; in some embodiments, specific binding is assessed by detecting or measuring the ability of the binding agent to compete for an alternative interaction between its partner and another entity. In some embodiments, specific binding is assessed by performing such detection or determination over a range of concentrations.

[0079] Specificity: The term "specificity" when used herein to refer to an agent with activity is understood by those skilled in the art to mean that the agent distinguishes between potential target entities or states. For example, in some embodiments, if an agent preferentially binds to its target in the presence of one or more competing alternative targets, the agent is said to "specifically" bind to its target. In many embodiments, specific interactions depend on the presence of specific structural features of the target entity (e.g., epitopes, clefts, binding sites). It should be understood that specificity does not have to be absolute. In some embodiments, specificity can be evaluated relative to the specificity of the binding agent for one or more other potential target entities (e.g., competitors). In some embodiments, specificity is evaluated relative to the specificity of a reference specific binding agent. In some embodiments, specificity is evaluated relative to the specificity of a reference nonspecific binding agent. In some embodiments, an agent or entity does not detectably bind to a competing alternative target under conditions of binding to its target entity. In some embodiments, a binding agent binds to its target entity with a higher association rate, a lower dissociation rate, increased affinity, reduced dissociation, and / or increased stability compared to a competing alternative target.

[0080] Specificity: As known in the art, "specificity" is a measure of the ability of a particular ligand to discriminate its binding partner from other potential binding partners.

[0081] Subject: As used herein, the term "subject" refers to an organism, typically a mammal (e.g., a human, including prenatal human forms in some embodiments). In some embodiments, the subject suffers from a relevant disease, condition, or illness. In some embodiments, the subject is susceptible to a disease, condition, or illness. In some embodiments, the subject exhibits one or more symptoms or characteristics of a disease, condition, or illness. In some embodiments, the subject does not exhibit any symptoms or characteristics of a disease, condition, or illness. In some embodiments, the subject is a person having one or more characteristics characterized by susceptibility to a disease, condition, or illness or risk of a disease, condition, or illness. In some embodiments, the subject is a patient. In some embodiments, the subject is an individual to whom a diagnosis and / or therapy is administered and / or has been administered.

[0082] Therapeutic agent: As used herein, the phrase "therapeutic agent" refers to an agent that has a therapeutic effect and / or elicits 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, alleviate, inhibit, prevent, delay the onset of, reduce the severity of, and / or reduce the incidence of one or more symptoms or features of a disease, disorder, and / or condition.

[0083] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" means an amount of a substance (e.g., a therapeutic agent, composition, and / or formulation) that elicits a desired biological response when administered as part of a treatment regimen. In some embodiments, a therapeutically effective amount of a substance is an amount that is 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 one of ordinary skill in the art, the effective amount of a substance may vary depending on factors such as the desired biological endpoint, the substance to be 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 alleviates, ameliorate, alleviate, inhibit, prevent, delay the onset, reduce the severity, and / or reduce the incidence of one or more symptoms or features 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.

[0084] Treatment: As used herein, the term "treatment" (also "treat" or "treating") refers to the administration of a therapy that partially or completely alleviates, ameliorates, relieves, suppresses, 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 directed to a subject who does not show signs of the relevant disease, disorder, and / or condition and / or to a subject who only shows early signs of the disease, disorder, and / or condition. Alternatively or additionally, such treatment may be directed to a subject who shows one or more established signs of the relevant disease, disorder, and / or condition. In some embodiments, treatment may be directed to a subject who has been diagnosed with the relevant disease, disorder, and / or condition. In some embodiments, treatment may be directed to a subject who is known to have one or more susceptibility factors that are statistically associated with an increased risk of developing the relevant disease, disorder, and / or condition. Thus, in some embodiments, treatment may be prophylactic; in some embodiments, treatment may be therapeutic.

[0085] 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, pre-metastatic, metastatic, and / or non-metastatic cells. In some embodiments, a tumor is associated with or is a manifestation of cancer. In some embodiments, a tumor may be a disseminated tumor or a liquid tumor. In some embodiments, a tumor may be a solid tumor.

[0086] Variant: As used herein in the context of molecules such as nucleic acids, proteins, or small molecules, the term "variant" refers to a molecule that exhibits significant structural identity with a reference molecule but differs structurally from the reference molecule, for example, in the presence or absence or level of one or more chemical moieties compared to the reference entity. In some embodiments, a variant is also functionally different from its reference molecule. Generally speaking, whether a particular molecule is appropriately considered a "variant" of a reference molecule is based on the degree of structural identity between the particular molecule and the reference molecule. As will be appreciated by those skilled in the art, any biological or chemical reference molecule has certain characteristic structural elements. By definition, a variant is a unique molecule that shares one or more such characteristic structural elements with a reference molecule but differs from it in at least one aspect. To give just a few examples, a polypeptide can have a characteristic sequence element composed of a plurality of amino acids having designated positions relative to each other in linear or three-dimensional space and / or contributing to a specific structural motif and / or biological function; a nucleic acid can have a characteristic sequence element composed of a plurality of nucleotide residues having designated positions relative to each other in linear or three-dimensional space. In some embodiments, variant polypeptides or nucleic acids may differ from reference polypeptides or nucleic acids due to one or more differences in amino acid or nucleotide sequence and / or one or more differences in a chemical moiety (e.g., carbohydrate, lipid, phosphate group), which is a covalent component of a polypeptide or nucleic acid (e.g., attached to a polypeptide or nucleic acid backbone). In some embodiments, variant polypeptides or nucleic acids exhibit an overall sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 99% with a reference polypeptide or nucleic acid. In some embodiments, variant polypeptides or nucleic acids do not share at least one characteristic sequence element with a reference polypeptide or nucleic acid. In some embodiments, a reference polypeptide or nucleic acid has one or more biological activities. In some embodiments, variant polypeptides or nucleic acids share one or more biological activities of a reference polypeptide or nucleic acid. In some embodiments, variant polypeptides or nucleic acids lack one or more biological activities of a reference polypeptide or nucleic acid. In some embodiments, variant polypeptides or nucleic acids exhibit reduced levels 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 that is identical to a reference polypeptide or nucleic acid but has a few sequence changes at specific positions. Typically, less than about 20%, about 15%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, or about 2% of the residues in the variant are substituted, inserted, or deleted compared to the reference. In some embodiments, the variant polypeptide or nucleic acid comprises about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, or about 1 substituted residue compared to the reference.Typically, a variant polypeptide or nucleic acid comprises a very small number (e.g., less than about 5, about 4, about 3, about 2, or about 1) of substituted, inserted, or deleted functional residues (i.e., residues involved in a specific biological activity) relative to a reference. In some embodiments, a variant polypeptide or nucleic acid comprises no more than about 5, about 4, about 3, about 2, or about 1 additions or deletions compared to a reference, and in some embodiments, no additions or deletions. In some embodiments, a variant polypeptide or nucleic acid comprises 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 a reference. In some embodiments, a reference polypeptide or nucleic acid is a polypeptide or nucleic acid that occurs in nature. In some embodiments, a reference polypeptide or nucleic acid is a human polypeptide or nucleic acid.

[0087] Vector: as used herein, refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is connected. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop to which additional DNA segments can be connected. Another type of vector is a viral vector, in which additional DNA segments can be connected to the viral genome. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (e.g., bacterial vectors and episomal mammalian vectors with bacterial replication origins). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of the host cell after being introduced into the host cell, thereby replicating together with the host genome. In addition, certain vectors are capable of directing the expression of genes operably connected to them. Such vectors are referred to herein as "expression vectors".

[0088] Viral inactivation or removal: As used herein, the term "viral inactivation or removal" describes 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 be derived from a source material (e.g., a host cell); alternatively or additionally, in some embodiments, the viruses present in the sample may have been introduced, for example, during processing of such source material. Those skilled in the art will appreciate that various techniques for achieving viral inactivation or removal, such as, for example, by pH inactivation, chemical inactivation (e.g., by using chemicals such as surfactants), etc. Those skilled in the art will understand that "pH viral inactivation" involves exposing viruses (e.g., a sample containing viruses) to a pH that inactivates (e.g., has been determined to inactivate) viruses.

[0089] 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) in a "normal" (as opposed to mutated, diseased, altered) state or background having a structure and / or activity found in nature. 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 (or forms) of a particular polypeptide or nucleic acid that is most commonly observed in a population (e.g., in a human population) is the "wild-type" form. DETAILED DESCRIPTION

[0090] The present disclosure particularly provides methods for producing, including purifying, phosphorylated forms of fusion polypeptides (e.g., as described in published international patent application WO2020 / 263399). Specifically, the present disclosure relates to producing phosphorylated forms of fusion polypeptides comprising (a) an immunomodulatory polypeptide comprising an immunoagonist portion; and (b) a metal hydroxide binding polypeptide whose amino acid sequence comprises multiple phosphorylation sites such that it can adopt phosphorylated and non-phosphorylated forms.

[0091] Fusion peptide

[0092] In some embodiments, fusion polypeptides according to the present disclosure can take on phosphorylated and non-phosphorylated forms.

[0093] Immunomodulatory peptides

[0094] In some embodiments, the fusion polypeptides of the present disclosure include at least one immunomodulatory polypeptide.

[0095] In some embodiments, the fusion polypeptide comprises two or more immunomodulatory polypeptides (e.g., two or more immunoagonist moieties). In some such embodiments, the fusion polypeptide comprises two or more identical immunomodulatory polypeptides; in some such embodiments, all immunomodulatory polypeptides in the fusion polypeptide according to the present disclosure are identical. In some such embodiments, the fusion polypeptide comprises two or more immunomodulatory polypeptides that are different from each other.

[0096] In some embodiments, the immunomodulatory polypeptide is or comprises at least one immunostimulatory moiety. In some embodiments, the immunostimulatory moiety is or comprises a functional fragment of a parent (e.g., wild-type) polypeptide; for example, in some embodiments, the immunomodulatory polypeptide is or comprises a functional fragment of a signaling-competent fragment. In some embodiments, the immunomodulatory polypeptide comprises one, two, three, four, five, or six immunostimulatory moieties.

[0097] Thus, in some embodiments, an immunomodulatory polypeptide may comprise more than one immunostimulatory moiety, which in various embodiments may be the same or different. In some such embodiments, two or more such immunostimulatory moieties are the same; in some embodiments, all such immunostimulatory moieties are the same. In some embodiments, an immunomodulatory polypeptide comprises two or more immunostimulatory moieties that are different from each other; in some embodiments, no two such immunostimulatory moieties are the same.

[0098] In some embodiments, the fusion polypeptide includes two or more immunomodulatory polypeptides (e.g., two or more immunoagonist portions) that include at least two immunomodulatory polypeptide (e.g., immunoagonist portion) subtypes—e.g., such that the fusion polypeptide includes at least two first subtypes and at least two second subtypes.

[0099] In some embodiments, an immunomodulatory polypeptide (eg, an immune agonist portion) activates or inhibits the activity of (eg, has signaling capability) a cell of the immune system.

[0100] For example, in some embodiments, signaling capability is characterized in that, when assessing binding to a specific binding partner, one or more immunoagonist portions or functional fragments thereof exhibit binding comparable to that of a reference standard (e.g., a wild-type polypeptide). For example, in some embodiments, signaling capability is characterized in that, when assessing a biological effect (e.g., in vitro or in vivo), one or more immunoagonist portions or functional fragments thereof exhibit a biological effect comparable to that of a reference standard (e.g., a wild-type polypeptide).

[0101] For example, in some embodiments, the immunomodulatory polypeptide (e.g., an immune agonist portion) is an immune response stimulating portion. In some embodiments, the response stimulating portion is, for example, but not limited to, a cytokine, a chemokine, an agonist antibody, an immune checkpoint inhibitor, or a combination thereof.

[0102] In some embodiments, the immunomodulatory polypeptide promotes differentiation and proliferation of immune cells (e.g., T cells).In some embodiments, the immunomodulatory polypeptide enhances the production of interferon gamma.

[0103] In some embodiments, the immunomodulatory polypeptide comprises an interleukin-12 (IL-12) immunomodulatory polypeptide (eg, an IL-12 immunoagonist portion).

[0104] IL-12 is a proinflammatory cytokine that plays an important role in innate and adaptive immunity. Wild-type IL-12 is a heterodimeric protein comprising two subunits, p35 (IL-12A; GenBenk GeneID: 3592) and p40 (IL-12B; GenBank GeneID: 3593), linked by a disulfide bond. 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 production and secretion of interferon gamma (IFN-γ).

[0105] 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 IL-35, and IL-12B can dimerize with p19 monomers to form the IL-12 family member IL23.

[0106] IL-12 plays an important role in innate and adaptive immune responses, and IL-12 dysregulation is associated with a variety of disease states. Exemplary such disease states include, but are not limited to, inflammatory bowel disease, psoriasis, diabetes, multiple sclerosis, rheumatoid arthritis, cancer, lupus erythematosus, primary biliary cholangitis, and Sjögren's syndrome ( syndrome) (Ullrich et al. EXCLI journal vol. 19, 1563-1589. Dec. 11, 2020). The use of IL-12 as a therapeutic modality has been widely studied, including for the treatment of tumors (Nastala CL et al. J Immunol. Aug. 15, 1994; Lasek et al. Cancer immunology, immunotherapy: CII vol. 63, 5 (2014): 419-35).

[0107] In some embodiments, the immunomodulatory polypeptide disclosed herein is or comprises an IL-12 immunoagonist portion. In some embodiments, the immunomodulatory polypeptide disclosed herein comprises multiple IL-12 immunoagonist portions. In some embodiments, the immunomodulatory polypeptide disclosed herein comprises exactly two IL-12 immunoagonist portions. In some embodiments, two or more IL-12 immunoagonist portions of multiple (e.g., two) IL-12 immunoagonist portions are identical portions. In some such embodiments, multiple (e.g., two) IL-12 immunoagonist portions are different portions. In some such embodiments, the IL-12 immunoagonist portion comprises an IL-12A polypeptide or a functional fragment thereof. In some embodiments, the IL-12 immunoagonist portion comprises an IL-12B polypeptide or a functional fragment thereof.

[0108] In some embodiments, the immunomodulatory polypeptides disclosed herein comprise an IL-12 immunoagonist portion comprising an IL-12A polypeptide or a functional fragment thereof and an IL-12 immunoagonist portion comprising an IL-12B polypeptide or a functional fragment thereof.

[0109] In some embodiments, the IL-12B immunoagonist portion is N-terminal to the IL-12A immunoagonist portion of the immunomodulatory polypeptide. In some embodiments, the IL-12A immunoagonist portion is N-terminal to the IL-12B immunoagonist portion of the immunomodulatory polypeptide.

[0110] In some embodiments, immunomodulatory polypeptides comprising multiple (e.g., two) IL-12 immunoagonist portions (e.g., IL-12A and / or IL-12B) are directly linked. In some embodiments, immunomodulatory polypeptides comprising multiple (e.g., two) IL-12 portions (e.g., IL-12A and / or IL-12B) are linked via a first linker. Non-limiting examples of linkers are discussed elsewhere herein.

[0111] In some embodiments, the immunomodulatory polypeptides disclosed herein comprise IL-12A and / or IL-12B immunoagonist portions comprising variants. In some embodiments, the IL-12A and / or IL-12B immunoagonist portion variants comprise substitutions, deletions, additions, and / or insertions relative to the wild-type IL-12A or IL-12B polynucleotide or amino acid sequence. In some embodiments, the IL-12A and / or IL-12B immunoagonist portion comprises multiple variants. In some embodiments, multiple variants comprise one or more substitutions, deletions, additions, and / or insertions relative to wild-type IL-12A or IL-12B. In some embodiments, the variants comprise substitutions that do not alter the amino acid sequence relative to wild-type IL-12A or IL-12B. In some embodiments, the IL12 variants comprise mutations.

[0112] In some embodiments, the immunomodulatory polypeptides disclosed herein comprise an IL-12A and / or IL-12B immunoagonist portion as a functional fragment thereof (e.g., a signaling-competent fragment). 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 a full-length IL-12A and a functional IL-12B fragment. In some embodiments, the immunomodulatory polypeptide comprises a full-length IL-12B and a functional IL-12A fragment.

[0113] In some embodiments, the 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 monomeric units (e.g., residues) as found in wild-type IL-12A or IL-12B.

[0114] In some embodiments, the immunomodulatory polypeptides disclosed herein comprise an IL-12A and / or IL-12B immunoagonist portion that is a human IL-12A and / or IL-12B immunoagonist portion.

[0115] 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, such as at least 85%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as 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, such as at least 85%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity to SEQ ID NO: 4. In some embodiments, an immunomodulatory polypeptide disclosed herein comprises an IL-12B immunoagonist portion having at least 80% sequence identity to SEQ ID NO:3 and an IL-12A immunoagonist portion having at least 80% sequence identity to SEQ ID NO:4.

[0116] 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%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity to SEQ ID NO:5.

[0117] Without being bound by a particular theory, replacement of hydroxyl groups (e.g., with phosphate groups) can increase adsorption of polypeptides via exchange with metal hydroxide (e.g., aluminum hydroxide) ligands and, further, can improve tumor retention and anti-tumor efficacy of such polypeptides (e.g., particularly fusion polypeptides comprising an immunomodulatory polypeptide and a metal hydroxide binding polypeptide in which such hydroxyl group replacement occurs).

[0118] In some embodiments, immunomodulatory polypeptides according to the present invention can be used in phosphorylated and non-phosphorylated forms. In some embodiments, the immunomodulatory polypeptide comprises at least one phosphorylated amino acid. In some embodiments, the immunomodulatory polypeptide comprises at least one kinase target motif. In some embodiments, the immunomodulatory polypeptide does not comprise a kinase target motif. In those embodiments, the immunomodulatory polypeptide may still comprise a phosphorylated amino acid. In some embodiments, immunomodulatory polypeptides comprising one or more phosphorylated amino acids facilitate strong binding of metals to metal hydroxides (e.g., aluminum hydroxide). Table 1 shows exemplary serine residues that can be phosphorylated in the immunomodulatory domain (e.g., S43, S154, S168, S233, S365, S398, S481 of SEQ ID NO. 2). In some embodiments, the immunomodulatory polypeptide comprises at least one phosphorylated serine. In some embodiments, the immunomodulatory polypeptide comprises at least two phosphorylated serine residues. In some embodiments, the immunomodulatory polypeptide comprises at least three phosphorylated serine residues. In some embodiments, the immunomodulatory polypeptide comprises at least four phosphorylated serine residues. In some embodiments, the immunomodulatory polypeptide comprises at least five phosphorylated serine residues, such as six serine residues, such as seven serine residues, such as eight serine residues, such as nine serine residues, such as ten serine residues.

[0119] Metal hydroxide binding peptides

[0120] In some embodiments, the fusion polypeptides of the present disclosure include at least one metal-binding polypeptide.

[0121] The present disclosure provides metal hydroxide binding polypeptides and fusion polypeptides comprising the same that exhibit high levels of adsorption to metal hydroxides and also exhibit desirable manufacturing characteristics (e.g., one or more of reproducibility, consistency, production of homogeneous phosphorylated fusion polypeptide preparations, etc.).

[0122] In some embodiments, the fusion polypeptide comprises two or more metal-binding polypeptides (e.g., two or more alum-binding polypeptides). In some embodiments, the fusion polypeptide comprises two or more identical metal-binding polypeptides; in some such embodiments, all metal-binding polypeptides in a fusion polypeptide according to the present disclosure are identical. In some such embodiments, the fusion polypeptide comprises two or more metal-binding polypeptides that are different from each other.

[0123] As discussed above, metal binding peptides can be fused to immunomodulatory polypeptides to achieve strong binding to metal hydroxides (such as aluminum hydroxide). Various immunomodulatory polypeptides can be fused to metal binding peptides. Without being bound by a particular theory, metal binding polypeptides adsorbed to alum in serum can be used to retain proteins and peptides in tumors.

[0124] In some embodiments, the metal hydroxide binding polypeptide comprises an amino acid sequence containing multiple phosphorylation sites, such that it can adopt phosphorylated and non-phosphorylated 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. Typically, the phosphorylated amino acid includes a hydroxyl group, such as serine (Ser, S), threonine (Thr, T), and tyrosine (Tyr, Y) residues. A kinase motif refers to an amino acid sequence that is adjacent to the N-terminus and / or C-terminus of an amino acid residue that can be phosphorylated. Without wishing to be bound by any one theory, many kinases comprise structural features that confer specificity, such that the kinase phosphorylates a specific amino acid (e.g., serine, threonine, or tyrosine) of a specific kinase target motif.

[0125] Depending on the specific type of kinase, the kinase target motif identified has a high degree of diversity. In some embodiments, the present disclosure provides a metal hydroxide binding polypeptide comprising one or more kinase target motifs of secretory pathway kinases. The secretory pathway as a cell secretes proteins and / or other biomolecules into the extracellular space refers to the endoplasmic reticulum (ER), Golgi apparatus (Golgi), cell membrane and lysosomal storage compartments and the vesicles moving therebetween. Secretory pathway kinases are located in the entire secretory pathway (e.g., in ER, Golgi apparatus, etc.) and play the role of phosphorylation secretory proteins (Sreelatha et al. Biochimica et biophysica acta vol. 1854, 10 Pt B (2015): 1687-93).

[0126] In some embodiments, the kinase of interest is a naturally occurring secretory pathway kinase (e.g., endogenously targeted to the secretory pathway for function). 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, tetrameric kinase 1, Fam20A, Fam20B, Fam20C, vertebrate orphan kinase (VLK), SGK196, and Fam69A, Fam69B, and Fam69C.

[0127] In some embodiments, the kinase of interest is a non-naturally occurring secretory pathway kinase. In some embodiments, the non-naturally occurring kinase is generated by linking a secretory signal peptide to a kinase that is endogenously localized to a non-secretory pathway cellular compartment.

[0128] In some embodiments, the kinase target motif is a target kinase motif of a secretory pathway kinase. In some embodiments, the secretory pathway kinase target kinase motif comprises a SXE motif. For example, Fam20C phosphorylates serine and has been shown to phosphorylate kinase target motifs comprising 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; Tagliabracci et al. (2015) Cell 161: 1619-1632; Tagliabracci et 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.

[0129] In some embodiments, the metal hydroxide binding polypeptide comprises at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve target kinase motifs. In some embodiments, the metal hydroxide binding polypeptide comprises at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve SXE motifs. In some embodiments, the metal hydroxide binding polypeptide comprises more than four 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.

[0130] In some embodiments, the metal hydroxide binding polypeptide is a metal hydroxide binding polypeptide whose amino acid sequence comprises a plurality of phosphorylation sites. In some embodiments, the plurality of phosphorylation sites comprises at least one, two, three, four, five, six, seven, eight, nine, ten, eleven or twelve target kinase motifs. In some embodiments, the plurality of phosphorylation sites comprises at least one, two, three, four, five, six, seven, eight, nine, ten, eleven or twelve SXE motifs. In some embodiments, the plurality of phosphorylation sites comprises more than four SXE motifs. In some embodiments, the plurality of phosphorylation sites comprises more than 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.

[0131] In some embodiments, at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve target kinase motifs (e.g., SXE motifs) are directly adjacent to (e.g., connected to) the next target kinase (e.g., SXE motif). In some embodiments, at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve target kinase motifs (e.g., SXE motifs) are separated (e.g., connected) from 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 comprising GGGEGGGG. In some embodiments, the spacer has an amino acid sequence comprising GGGGG. In some embodiments, the spacer has an amino acid sequence comprising GGGG.

[0132] 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.

[0133] In some embodiments, the metal hydroxide binding polypeptide comprises a terminator sequence (e.g., an amino acid sequence) at the c-terminus of the fusion polypeptide. In some embodiments, the terminator sequence comprises a plurality of amino acid residues. In some embodiments, the plurality of amino acid residues comprises GGGG. In some such embodiments, the terminator sequence comprises the amino acid sequence GGGGS.

[0134] In some embodiments, the desired (e.g., optimal) number of kinase target motifs and / or kinase motif spacing can be determined based on, for example, one or more of the desired phosphate content to achieve strong metal hydroxide retention and / or avoid one or more manufacturing challenges (e.g., the present disclosure understands that it can be associated with highly phosphorylated elements). In some embodiments, the desired (e.g., optimal) number of kinase motifs and / or spacing results in exposure of the polypeptide to the kinase to achieve the desired level of fusion polypeptide phosphorylation. In some embodiments, the improved fusion polypeptides as described herein result in one or more improvements in repeatability, consistency, and / or production of homogeneous phosphorylated fusion polypeptides. For example, in some embodiments, the provided technology enables the reproducible manufacture of comparable preparations (e.g., preparations 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.

[0135] In some embodiments, the degree of phosphorylation (e.g., the 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. 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.5, 11, 11.5, or 12.

[0136] connector

[0137] In some embodiments, a fusion polypeptide as described herein may comprise one or more linkers and / or spacers.

[0138] For example, in some embodiments, the fusion polypeptide comprises an immunomodulatory polypeptide comprising a first immunoagonist portion and a second immunoagonist portion. In some embodiments, the first immunoagonist portion and the second immunoagonist portion are connected via a first linker.

[0139] In some embodiments, the fusion polypeptides of the present disclosure include an immunomodulatory polypeptide and a metal hydroxide binding polypeptide. In some embodiments, the immunomodulatory polypeptide and the metal hydroxide binding polypeptide are linked via a second linker.

[0140] 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 comprise a non-naturally occurring polypeptide that is a modified form of a naturally occurring polypeptide.

[0141] In some embodiments, the length of the polypeptide linker of the present disclosure is at least one amino acid and can be any suitable number of amino acids. In some embodiments, the length of the polypeptide linker is 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.

[0142] 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 comprises (Gly4Ser) n wherein 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 .

[0143] 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.

[0144] Variants

[0145] In some embodiments, the immunomodulatory polypeptides or metal hydroxide binding polypeptides utilized in accordance with the present disclosure are variants of a related reference polypeptide (eg, a wild-type polypeptide or a functional portion thereof).

[0146] In some embodiments, the variant exhibits at least 70% identity to its reference polypeptide. In some such embodiments, the variant exhibits at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to its reference polypeptide.

[0147] In some embodiments, variant comprises one or more conservative or other non-destructive modifications (e.g., substitutions, deletions or additions) relative to its reference. In some embodiments, variant does not contain any destructive modifications (e.g., substitutions, deletions or additions) such that the immunomodulatory polypeptide maintains one or more functional characteristics of reference. In some embodiments, maintenance means that the immunomodulatory polypeptide exhibits comparable activity (e.g., signal transduction capability or combination) compared to an appropriate reference standard (e.g., wild-type immunomodulatory polypeptide). For example, in some such embodiments, the immunomodulatory polypeptide maintains at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher activity compared to an appropriate reference standard (e.g., wild-type immunomodulatory polypeptide).

[0148] Metal hydroxides

[0149] 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 the metal hydroxide when exposed to the metal hydroxide. The phosphorylated fusion polypeptide forms a complex via adsorption to the metal hydroxide. Without wishing to be bound by any one theory, it is assumed that the phosphorylated fusion polypeptide is adsorbed to the metal hydroxide by ligand exchange. For example, ligand exchange is to replace or exchange surface hydroxyls by another ligand. In some embodiments, substitution or exchange of surface hydroxyls occurs by hydroxyl alternative groups (e.g., phosphate groups).

[0150] In some embodiments, the metal hydroxide is a substance comprising at least one hydroxyl group that binds to a metal. According to the present disclosure, in some embodiments, the metal hydroxide can adsorb a fusion polypeptide comprising a hydroxyl replacement moiety. In some embodiments, the hydroxyl replacement moiety is a phosphate group.

[0151] In some embodiments, the metal hydroxide is selected based on its inherent qualities or characteristics. In some embodiments, the metal hydroxide is selected due to its biocompatibility for use 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 ferric hydroxide. Those skilled in the art will recognize that any number of metal hydroxides can be successfully utilized in accordance with the present disclosure.

[0152] Phosphorylated fusion peptide preparations

[0153] Among other things, the present disclosure provides preparations of fusion polypeptides, and in particular preparations of phosphorylated fusion polypeptides, having particularly desirable characteristics (e.g., the level of purity and / or the extent of phosphorylation of such phosphorylated fusion polypeptides in the resulting preparation, such as the average extent of phosphorylation of the fusion polypeptide and / or the distribution of specific phospho-forms).

[0154] Among other things, the present disclosure identifies sources of problems with certain methods of making related fusion polypeptides, including, for example, their ability to produce preparations with an appropriate degree and / or consistency (e.g., homogeneity) of phosphorylation and / or their ability to produce preparations of sufficient purity. The present disclosure provides particularly high-purity preparations of phosphorylated fusion polypeptides, including high-purity preparations characterized by a particular degree and / or consistency of phosphorylation.

[0155] Without wishing to be bound by any particular theory, the present disclosure indicates that excessively high or low levels of phosphorylation can negatively impact the activity of fusion polypeptide preparations.

[0156] The present disclosure provides particularly useful methods for manufacturing phosphorylated forms of fusion polypeptides. In some embodiments, the present disclosure provides methods for manufacturing phosphorylated forms of fusion polypeptides disclosed herein by contacting the fusion polypeptide with a kinase. Therefore, the manufacturing methods of the present invention as described herein provide certain fusion polypeptide preparations characterized by having a particularly desired degree of phosphorylation. Alternatively or additionally, in some embodiments, the technology provided achieves the manufacture of desired preparations as described herein (e.g., with phosphorylation levels and / or consistency and / or purity levels as described herein) in a particularly high yield. In some embodiments, the present invention provides methods for manufacturing high-purity preparations of phosphorylated forms of fusion polypeptides.

[0157] In some embodiments, the highly pure phosphorylated fusion polypeptide preparation comprises more phosphorylated fusion polypeptide relative to the non-phosphorylated fusion polypeptide. In some embodiments, the highly pure phosphorylated fusion polypeptide preparation comprises primarily phosphorylated fusion polypeptide relative to the non-phosphorylated fusion polypeptide. In some embodiments, the highly pure phosphorylated fusion polypeptide preparation comprises at least 90% phosphorylated fusion polypeptide, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% phosphorylated fusion polypeptide.

[0158] In some embodiments, the fusion polypeptide preparation comprises a non-phosphorylated 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 non-phosphorylated form and the phosphorylated form of the fusion polypeptide.

[0159] In some embodiments, a fusion polypeptide formulation is a formulation comprising a fusion polypeptide in which one or more characterizing attributes are assessed 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 heterogeneity of phosphorylation.

[0160] In some embodiments, exogenous expression techniques are used to produce the fusion polypeptides described herein having phosphorylated and non-phosphorylated forms in host cells.

[0161] In some embodiments, the fusion polypeptide preparation includes a phosphorylated fusion polypeptide. In some embodiments, the fusion polypeptide preparation is a high-purity preparation of the phosphorylated form of the fusion polypeptide. In some embodiments, the fusion polypeptide preparation comprises a mixture of the non-phosphorylated form and the phosphorylated form of the fusion polypeptide. In some embodiments, the fusion polypeptide preparation comprises more phosphorylated fusion polypeptide than the non-phosphorylated fusion polypeptide. In some embodiments, the phosphorylated fusion polypeptide preparation includes a fusion polypeptide with different degrees of phosphorylation as described above. In some embodiments, the fusion polypeptide preparation is prepared in a Tris buffer of about pH 7 to about pH 8. In some embodiments, the fusion polypeptide preparation comprises a salt (e.g., NaCl).

[0162] Production of phosphorylated fusion peptides

[0163] Expression in host cells

[0164] In some embodiments of the present disclosure, the fusion polypeptide is produced by producing it in a host cell (such as a mammalian cell). Typically, such host cells (e.g., such mammalian cells) will be engineered to express the fusion polypeptide. Those skilled in the art are familiar with various techniques for introducing exogenous genetic sequences (e.g., encoding fusion polypeptides and / or kinases) into host cells (such as mammalian host cells) for their expression.

[0165] For example, in some embodiments, a polynucleotide (e.g., DNA or RNA) encoding a fusion polypeptide of the present invention can be prepared, for example, for introduction into a host cell. For example, the sequence encoding the fusion polypeptide can be excised from the DNA using a restriction enzyme, can be amplified from a plasmid or genomic polynucleotide sequence using, for example, the polymerase chain reaction, or can be synthesized using chemical synthesis techniques. In some embodiments, a combination of known methods is used to prepare a recombinant polynucleotide encoding a fusion polypeptide of the present invention.

[0166] The recombinant polynucleotide encoding the fusion polypeptide of the present invention can be cloned into a vector capable of expressing the fusion polypeptide. Cloning can be carried out according to various available methods (e.g., Gibson assembly, restriction digestion, and ligation). 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.

[0167] In some embodiments, the vector capable of expression comprises a recombinant polynucleotide encoding a fusion polypeptide of the present invention, which is operably connected to one or more sequences (e.g., promoters, start signals, stop signals, polyadenylation signals, activators, repressors, etc.) regulating and controlling polynucleotide expression. In some embodiments, one or more regulatory sequences for control expression are selected to achieve desired expression levels. In some embodiments, the sequence (e.g., promoter) for controlling expression is utilized. In some embodiments, the sequence (e.g., promoter) for controlling expression is utilized to achieve desired expression levels of a plurality of recombinant polynucleotides encoding a plurality of polypeptides. In some embodiments, a plurality of recombinant polypeptides are expressed by the same vector (e.g., bicistronic vector, tricistronic vector, polycistronic vector). In some embodiments, a plurality of recombinant polypeptides are expressed, each of the polypeptides being expressed by a separate vector.

[0168] In some embodiments, an expression-capable vector comprising a recombinant polynucleotide encoding a fusion polypeptide of the present disclosure is used to express the fusion polypeptide in a host cell.

[0169] The host cell can be selected from various available and known host cells suitable for expressing the fusion polypeptides disclosed herein (eg, human embryonic kidney (HEK) cells, suspension HEK293 cells, Chinese Hamster Ovary cells).

[0170] Various methods are available for introducing vectors into host cells. In some embodiments, transfection can be used to introduce vectors into host cells. In some embodiments, transfection is accomplished, for example, using calcium phosphate transfection, lipofection, or polyethyleneimine-mediated transfection. In some embodiments, transduction can be used to introduce vectors into host cells.

[0171] In some embodiments, host cells (eg, producer cells) are used to produce the phosphorylated form of the fusion polypeptide.

[0172] In some embodiments, host cells expressing fusion polypeptides and / or kinases 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, host cells are cultured at a temperature in the range of 30° C. to 40° C. In some embodiments, the temperature is lower (e.g., 33° C.) during the production phase. In some embodiments, cell extracts are harvested through 2 or 3 stage filters, followed by terminal sterilization 0.22 μm filtration.

[0173] 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 a host cell such that the host cell expresses the kinase. In many embodiments, as described herein, a nucleic acid encoding a fusion polypeptide and a nucleic acid encoding a kinase that phosphorylates the fusion polypeptide are introduced into the same host cell; in some such embodiments, a single nucleic acid molecule can encode both.

[0174] In some embodiments, the nucleic acid molecule introduced into the cell is RNA (e.g., mRNA); in some such embodiments, the encoded polypeptide (e.g., fusion polypeptide and / or kinase) is 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 (e.g., fusion polypeptide and / or kinase) is expressed therefrom,

[0175] In some embodiments, the nucleic acid molecule (e.g., a nucleic acid molecule encoding a fusion polypeptide and / or a kinase) introduced into a cell comprises one or more expression elements, e.g., that can regulate the expression of such encoded polypeptides. Alternatively or additionally, in some embodiments, the nucleic acid molecule (e.g., a nucleic acid molecule encoding a fusion polypeptide and / or a kinase) introduced into a cell can be designed or intended to associate (e.g., by integration) with one or more regulatory elements in the host cell.

[0176] In some embodiments, a vector (eg, a transposon) comprising a sequence encoding a fusion polypeptide and / or kinase as described herein is used to express the fusion polypeptide and / or kinase in a host cell.

[0177] In some embodiments, the host cell can be selected from various available and known host cells suitable for expressing the fusion polypeptides disclosed herein (e.g., human embryonic kidney (HEK) cells, suspension HEK293 cells, Chinese hamster ovary cells). In some embodiments, the host cell is a mammalian cell.

[0178] Various methods can be obtained to introduce nucleic acid (e.g., vector, such as expression vector) into a host cell. In some embodiments, transfection can be used to introduce nucleic acid into a host cell. In some embodiments, transfection is completed using calcium phosphate transfection, lipofection, or polyethyleneimine-mediated transfection. In some embodiments, transduction can be used to introduce nucleic acid into a host cell. In some embodiments, electroporation can be used to introduce nucleic acid into a host cell. In some embodiments, particles can be used to deliver, such as polymer particles, lipid particles, gold particles, etc., to introduce nucleic acid into a host cell.

[0179] Phosphorylation

[0180] In some embodiments, the present disclosure provides a method for manufacturing a phosphorylated form of a fusion polypeptide disclosed herein by contacting the fusion polypeptide with a kinase. In some embodiments, a nucleic acid (such as a nucleic acid encoding the fusion polypeptide and / or kinase) is introduced into a host cell. In some embodiments, the fusion polypeptide is contacted with the kinase by co-expressing the fusion polypeptide and the kinase in the host cell. In some embodiments, co-expression is achieved by introducing two vectors into the host cell, one vector comprising a recombinant polynucleotide encoding the fusion polypeptide, and the other vector comprising a recombinant polynucleotide encoding the kinase. In some embodiments, co-expression is achieved by introducing a single polycistronic (e.g., bicistronic) vector comprising multiple recombinant polynucleotides (e.g., such as a transposon). In some embodiments, the recombinant polynucleotide encodes the fusion polypeptide and the recombinant polynucleotide encodes the kinase. In some embodiments, the transformed host cell is cultured after the vector (e.g., a transposon) is introduced into the host cell. Without wishing to be bound by any one theory, after the fusion polypeptide and the kinase are co-expressed in the host cell, the kinase can contact the fusion polypeptide and phosphorylate it.

[0181] In some embodiments, co-expression is achieved by introducing two vectors into a host cell, one vector comprising a recombinant polynucleotide encoding a fusion polypeptide, and the other vector comprising a recombinant polynucleotide encoding a kinase. In some embodiments, two vectors are introduced into a host cell at a ratio of a vector encoding a fusion polypeptide to a vector encoding a kinase, the ratio being optimized to achieve the desired relative expression level of the fusion polypeptide to the kinase. In some embodiments, the ratio of the vector encoding the fusion polypeptide to the 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.

[0182] In some embodiments, coexpression is achieved by introducing a vector (e.g., bicistronic vector) comprising a recombinant polynucleotide encoding a fusion polypeptide and a recombinant polypeptide encoding a kinase into a host cell. In some embodiments, coexpression is achieved by introducing a transposon comprising a recombinant polynucleotide encoding a fusion polypeptide and a recombinant polypeptide encoding a kinase into a host cell. In some embodiments, the transposon is a DNA transposon. In some embodiments, the transposon or a portion thereof (e.g., comprising nucleotides encoding a fusion polypeptide and nucleotides encoding a kinase) is integrated into the host cell genome by integrase (integration enzyme) (i.e., by integrase (integrase enzyme), such as DDE / D integrase). In some embodiments, 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 the polynucleotide encoding the fusion polypeptide of the present disclosure is integrated into specific multiple host cell genome loci. In some embodiments, the integration of the polynucleotide encoding the fusion polypeptide of the present disclosure is irreversible. Fusion polypeptide is irreversibly integrated into the host cell genome to ensure stable integration. Thus allowing the production of highly stable cell lines. In some embodiments, the recombinant polynucleotide encoding the fusion polypeptide and the recombinant polynucleotide encoding the kinase are operably connected to one or more sequences (e.g., promoter, start signal, stop signal, polyadenylation signal, activator, repressor, etc.) that control expression. In some embodiments, the one or more sequences selected for control expression are to achieve desired expression levels. In some embodiments, the desired ratio of the expression of fusion polypeptide and kinase is achieved using a plurality of regulatory nucleotide sequences (e.g., promoter) that control expression. 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 single transposon with two promoters is used to express the fusion polypeptide and kinase to achieve a specific ratio (e.g., 2: 1, 4: 1, 8: 1 or 15: 1). In some embodiments, a single, separate transposon comprises promoters of varying strengths to produce 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 SV40 or Ubc promoter. In some embodiments, the kinase is controlled by the SV40 promoter or Ubc promoter. In some embodiments, the ratio of fusion polypeptide to kinase is 8:1.

[0183] In some embodiments, the transformed host cell (i.e., the host cell into which the nucleic acid (such as nucleic acid encoding fusion polypeptide and / or kinase) enters) is cultured after such transformation, for example to allow expression of the recombinant polynucleotide. In some embodiments, the transformed host cell is cultured for at least 12 hours, 16 hours, 20 hours, 24 hours, 28 hours, 32 hours, 36 hours, 40 hours, 44 hours, 48 hours, 52 hours, 56 hours, 60 hours, 64 hours, 68 hours, 72 hours or longer. In some embodiments, the transformed host cell is cultured for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days or longer. The transformed host cell is cultured under the growth conditions (e.g., temperature, carbon dioxide level, growth medium) according to the requirement of the selected host cell. Those skilled in the art will recognize that the culture conditions of the selected host cell are well known in the art. In some embodiments, the host cell secretes the phosphorylated form of the fusion polypeptide into the cell extract. In some embodiments, the host cell may show increased secretion of the phosphorylated fusion polypeptide into the cell extract compared to the non-phosphorylated fusion polypeptide. In some embodiments, the host cell secretes comparable levels of phosphorylated fusion polypeptide 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 the non-phosphorylated form and the phosphorylated form of the fusion polypeptide. In some embodiments, the cell extract comprises more phosphorylated form of the fusion polypeptide than the non-phosphorylated form of the fusion polypeptide.

[0184] In some embodiments, the host cell secretes the phosphorylated form of the fusion polypeptide into the cell extract, but does not secrete the kinase. In some embodiments, the host cell does not secrete the kinase or secretes only a small amount of the kinase.

[0185] 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.

[0186] In some embodiments, the phosphorylated form of the fusion polypeptide is harvested from the transformed host cells and clarified by centrifugation.

[0187] In some embodiments, the transformed host cell is characterized in that its culture produces the fusion protein with a titer of at least 200 mg / L, such as at least 250 mg / L, such as at least 300 mg / L, such as at least 350 mg / L, such as at least 400 mg / L, such as at least 450 mg / L, such as at least 500 mg / L, such as at least 550 mg / L, such as at least 600 mg / L, such as at least 650 mg / L, such as at least 700 mg / L, such as at least 750 mg / L, such as at least 800 mg / L, such as at least 850 mg / L, such as at least 900 mg / L, such as at least 950 mg / L, such as at least 1 g / L or more.

[0188] In some embodiments, one or more serine residues at positions 43, 281, 306, 311, 316, 365, or 481 of SEQ ID NO: 2 are phosphorylated. In some embodiments, one or more serine residues at positions 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 positions 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 positions 43, 154, 168, 281, 306, 311, 316, 365, 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.

[0189] purification

[0190] In some embodiments, the present disclosure provides techniques (e.g., manufacturing techniques) that are or include purification methods (e.g., methods including one or more purification steps). In some embodiments, the phosphorylated form of the fusion protein is purified from a cell extract described herein.

[0191] In some embodiments, purification steps can include removing common undesirable products (eg, residual proteins, host cell contaminants (eg, host DNA and / or proteins, etc.)) from the cell extract.

[0192] In some embodiments, the manufacturing method including one or more purification steps results in a high-purity preparation of the phosphorylated form of the fusion polypeptide (see, for example, Examples 10-11). In some embodiments, the high-purity preparation of the phosphorylated form of the fusion polypeptide comprises host cell proteins comparable to the reduction of the cell extract described above. In some embodiments, such high-purity preparations do not comprise any host cell proteins. In some embodiments, the high-purity preparation comprises less than 100ng / mg host cell protein, such as less than 50ng / mg, such as less than 40ng / mg, such as less than 30ng / mg, such as less than 20ng / mg, such as less than 10ng / mg, such as less than 9ng / mg, such as less than 8.5ng / mg host cell protein. In some embodiments, the high-purity preparation of the phosphorylated form of the fusion polypeptide comprises host cell DNA comparable to the reduction of the cell extract described above. In some embodiments, such high-purity preparations do not comprise any host cell DNA. In some embodiments, the highly pure preparation comprises less than 10 pg / mg of host cell DNA, such as less than 9 pg / mg, such as less than 8 pg / mg, such as less than 7 pg / mg, such as less than 6 pg / mg, such as less than 5 pg / mg, such as less than 4 pg / mg, such as less than 3 pg / mg, such as less than 2 pg / mg, such as less than 1.5 pg / mg, such as less than 1 pg / mg, such as less than 0.9 pg / mg, such as less than 0.8 pg / mg, such as less than 0.7 pg / mg of host cell DNA.

[0193] In some embodiments, the high-purity formulation contains a low level of in-process compounds. In some embodiments, the in-process compound can be tropolone, pluronic, PDMS, octamethylcyclotetrasiloxane D4, TDAO, and / or Fam20C. In some embodiments, the high-purity formulation contains less than 1 mg / mL TDAO, such as less than 0.9 mg / mL TDAO, such as less than 0.8 mg / mL TDAO, such as less than 0.7 mg / mL TDAO, such as less than 0.6 mg / mL TDAO, such as less than 0.5 mg / mL TDAO, such as less than 0.4 mg / mL TDAO, such as less than 0.3 mg / mL TDAO. In some embodiments, the highly pure preparation comprises less than 5000 ng Fam20C / mg IL-12 fusion polypeptide, such as less than 4000 ng Fam20C / mg IL-12 fusion polypeptide, such as less than 3000 ng Fam20C / mg IL-12 fusion polypeptide, such as less than 2500 ng Fam20C / mg IL-12 fusion polypeptide, such as less than 2000 ng Fam20C / mg IL-12 fusion polypeptide, such as less than 1800 ng Fam20C / mg IL-12 fusion polypeptide, such as less than 1000 ng Fam20C / mg IL-12 fusion polypeptide, such as less than 750 ng Fam20C / mg IL-12 fusion polypeptide, such as less than 500 ng Fam20C / mg IL-12 fusion polypeptide, such as less than 300 ng Fam20C / mg IL-12 fusion polypeptide, such as less than 200 ng Fam20C / mg IL-12 fusion polypeptide, such as less than 100 ng Fam20C / mg IL-12 fusion polypeptide, such as less than 80 ng Fam20C / mg IL-12 fusion polypeptide, such as less than 70 ng Fam20C / mg IL-12 fusion polypeptide, such as less than 60 ng Fam20C / mg IL-12 fusion polypeptide.

[0194] In some embodiments, the phosphorylated form of the fusion polypeptide can be purified by including one or more chromatography purification steps. In some embodiments, one or more conventional chromatography steps are used. In some embodiments, conventional chromatography steps utilize anion or cation exchange, hydrophobic interaction, or hydroxyapatite chromatography.

[0195] In some embodiments, the phosphorylated form of the fusion polypeptide can be purified by one or more purification steps selected from an ion chromatography step (e.g., an anion chromatography step) and a hydrophobic interaction chromatography step, thereby resulting in separation of the phosphorylated fusion polypeptide from impurities.

[0196] Those skilled in the art will be familiar with various purification (e.g., chromatography) substrates and formats thereof that can be utilized in accordance with the present disclosure. For example, in some embodiments, beads, particles, microspheres, resins, etc. can be utilized. In some embodiments, the substrate used for purification (e.g., for chromatography) has properties that allow the fusion polypeptide to have a different retention time relative to any other undesirable components in the fusion polypeptide preparation in accordance with the present disclosure.

[0197] In some embodiments, the phosphorylated form of the fusion polypeptide is not purified by affinity-based purification methods. For example, in some embodiments, the provided purification techniques do not utilize affinity chromatography.

[0198] In some embodiments, the phosphorylated form of the fusion polypeptide can be eluted from a solid substrate. In some embodiments, specific elution can be used to elute. For example, in some embodiments, specific elution is accomplished by attacking the polypeptide-substrate complex with one or more agents that will complete a complex with the substrate or polypeptide, releasing the polypeptide into the solution. In some embodiments, non-specific elution can be used to elute. For example, in some embodiments, non-specific elution is accomplished by manipulating solvent or buffer conditions (e.g., increasing the concentration of a buffer such as imidazole buffer) to reduce the association rate constant, causing the polypeptide to dissociate from the substrate, thereby completing non-specific elution.

[0199] First chromatographic step

[0200] In some embodiments, methods according to the present disclosure comprise at least one ion chromatography step (eg, an anion chromatography step). In some embodiments, methods according to the present disclosure comprise at least one anion chromatography step.

[0201] 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 one theory, polypeptide phosphorylation imparts charge variability to the polypeptide, thereby allowing the use of ion exchange chromatography (e.g., anion exchange chromatography) to separate differentially phosphorylated polypeptides. Anion exchange chromatography is a form of ion exchange in which negatively charged biomolecules (e.g., phosphorylated forms of the fusion polypeptides disclosed herein) are bound to a positively charged solid substrate (e.g., a resin). The positively charged solid substrate thus captures the negatively charged fusion polypeptide from the cell extract and simultaneously removes positively charged impurities and fusion polypeptide aggregates from the cell extract because the aggregates appear to have less negative charge.

[0202] In some embodiments, anion exchange chromatography can be used to distinguish polypeptides having different numbers of phosphorylated amino acid residues (e.g., differentially phosphorylated polypeptides). Anion exchange chromatography can enrich fusion polypeptide preparations for highly phosphorylated species (e.g., fusion polypeptides having more than 6 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 produced by using an anion exchange chromatography step (e.g., as a first step).

[0203] In some embodiments, the step of 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.

[0204] In some embodiments, anion exchange chromatography utilizes ion exchange resins with covalently bound positively charged groups (such as quaternary amino groups). Commercially available anion exchange resins include Q agarose, DEAE agarose, TMAE, GigaCap Q 650M and 650S. In some embodiments, negatively charged biomolecules (e.g., phosphorylated forms of fusion polypeptides) are bound to anion exchange materials and include: under appropriate conditions (e.g., pH / conductivity), negatively charged biomolecules are exposed to resin, thereby immobilizing the biomolecules to the anion exchange resin through ionic interactions between the negatively charged biomolecules and the ion exchange material, one or more charged groups.

[0205] The washing step may entail 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 include different conditions, such as pH and conductivity, in order to dissociate impurities that are non-specifically bound to the chromatography resin. In some embodiments, the washing step utilizes a mixture of an equilibration buffer and an elution buffer.

[0206] The phosphorylated form of the fusion polypeptide can be eluted from the solid substrate (e.g., a positively charged resin) using an eluent. 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 salt concentration and / or a different pH to promote the dissociation of the negatively charged agent from the chromatography resin.

[0207] 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 different phosphorylated polypeptides (i.e., separation of different phospho-forms can be achieved).

[0208] In some embodiments, buffer is for example Tris buffer.In some embodiments, utilize the linear gradient of Tris buffer.In some embodiments, the linear gradient of Tris buffer is included in the linear gradient from 20mMTris, pH 7.1 to 20mM Tris, 1M NaCl, pH 7.1 in the predetermined time period.In some embodiments, linear gradient carries out 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 the period of longer time.In some embodiments, the first anion chromatography capture step uses Tris buffer.In some embodiments, the first anion chromatography capture step is carried out under the pH of approximately 6 to approximately 9 (such as approximately 7 to approximately 8).

[0209] In some embodiments, the first anion chromatography capture step is performed using capture beads. In some embodiments, the first capture bead has a diameter of at least 50 μm, such as at least 55 μm, such as at least 60 μm, such as at least 65 μm, such as at least 70 μm, such as at least 75 μm. In some embodiments, the first capture bead is a GigaCap Q 650M.

[0210] In some embodiments, the first anion chromatography capture step includes a fixation step (e.g., phosphorylation fusion polypeptide is bound to a chromatographic column), a pre-elution washing step, and an elution step (e.g., eluting the phosphorylation fusion polypeptide). In some embodiments, resin beads having a particle size of about 50 to about 100 microns (mean value), such as 75 microns (mean value) are used to fix the phosphorylation fusion polypeptide. In some embodiments, a fixed composition (e.g., using 0M sodium chloride) is used during the phosphorylation fusion polypeptide is fixed to the chromatographic column. In some embodiments, a pre-elution composition (e.g., using 215mM sodium chloride) having an intermediate salt concentration comparable to that of the fixed composition and the elution composition is used during the pre-elution washing step. In some embodiments, an elution composition (e.g., using 350mM sodium chloride) is used during the phosphorylation fusion polypeptide is eluted from the chromatographic column. 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 200-400 cm / h is used (eg, 300 cm / h).

[0211] Second chromatographic step

[0212] 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 carried out after the first anion chromatography step. Without being bound by a particular theory, the phosphorylated form of the fusion protein according to the present disclosure has low hydrophobicity (for example, due to its phosphorylation degree and therefore its charge), which can be used to separate it from hydrophobic impurities. In some embodiments, hydrophobic host cell impurities (for example, host cell proteins) and / or fusion polypeptide aggregates are separated from the preparation of the phosphorylated form of the fusion polypeptide. In some embodiments, the preparation (for example, high purity preparation) comprises less than 5% aggregation fusion polypeptide, such as less than 4%, such as less than 3%, such as less than 3%, such as less than 2%, such as less than 1%, such as less than 0.9%, such as less than 0.8% aggregation fusion polypeptide.

[0213] In some embodiments, the hydrophobic interaction step can separate product or process-related impurities (e.g., host cell proteins or aggregated products (e.g., fusion polypeptide aggregates)) from the phosphorylated form of the fusion polypeptide based on the differences in the hydrophobic interactions of the phosphorylated fusion polypeptide and the impurities with the hydrophobic material. In some embodiments, such a step can be referred to as a polishing step.

[0214] Examples of hydrophobic interaction resins include, but are not limited to, hydrophobic ligands, such as alkyl groups or aryl groups such as phenyl groups having 2 to 8 carbon atoms. In some embodiments, a negatively charged agent (e.g., a phosphorylated form of a fusion polypeptide) is bound to a hydrophobic interaction resin comprising exposing the biomolecule to the resin under appropriate conditions (pH / conductivity), thereby immobilizing the biomolecule to the hydrophobic resin by the hydrophobic interaction between the biomolecule and the non-polar group of the hydrophobic interaction material. Hydrophobic interaction binding typically occurs under high salt concentrations (e.g., 1 to 1.8 M ammonium sulfate). In some embodiments, the phosphorylated form of the fusion polypeptide is fixed under 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 from 1.4 M ammonium sulfate to 0 M ammonium sulfate.

[0215] The washing step may entail passing an appropriate buffer through the chromatography resin to wash away unwanted materials, such as less hydrophobic host cell proteins. In some embodiments, the wash buffer may have a different pH to promote the dissociation of the less hydrophobic agent from impurities that are non-specifically bound to the chromatography resin. In some embodiments, the washing step utilizes a mixture of an equilibration buffer and an elution buffer.

[0216] The phosphorylated form of the fusion polypeptide can be eluted from a solid substrate (e.g., a hydrophobic resin) using an eluent. In some embodiments, a buffer that reduces the interaction between the hydrophobic interaction resin and the negatively charged agent (e.g., phosphorylated fusion polypeptide) is used to elute the low hydrophobic agent (e.g., the phosphorylated form of the fusion polypeptide) from the hydrophobic resin. In some embodiments, such an elution buffer may have a lower salt concentration or pH change that promotes the dissociation of the biomolecule from the chromatography resin. In some embodiments, the phosphorylated form of the fusion polypeptide is eluted at a low salt concentration (e.g., 750 mM sodium sulfate).

[0217] In some embodiments, the hydrophobic interaction chromatography step includes a fixing step (e.g., binding the phosphorylated fusion polypeptide to a chromatographic column) and an elution step (e.g., eluting the phosphorylated fusion polypeptide). In some embodiments, a resin bead having a particle size of about 50 to about 100 microns (average value), such as 75 microns (average value) is used to fix the phosphorylated fusion polypeptide. In some embodiments, a fixed composition with a high salt concentration is used during the phosphorylated fusion polypeptide is fixed to the chromatographic column (e.g., using 1.4M ammonium sulfate). In some embodiments, an elution composition with a low salt concentration is used during the phosphorylated fusion polypeptide is eluted from the chromatographic column (e.g., using 740mM ammonium sulfate). In some embodiments, the hydrophobic interaction chromatography 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 200-350cm / h (e.g., 275cm / h) is used.

[0218] The third chromatographic step

[0219] In some embodiments, the method according to the present disclosure comprises a first anion chromatography step and a second anion chromatography step. In some embodiments, the method according to the present disclosure comprises 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.

[0220] In some embodiments, the methods according to the present disclosure include the following steps:

[0221] i) a first anion chromatography step;

[0222] ii) a hydrophobic interaction chromatography step; and

[0223] iii) Second anion chromatography step.

[0224] In some embodiments, the second anion chromatography step is performed using capture beads. In some embodiments, the diameter of the second step capture beads is at most 50 μm, at most 45 μm, at most 40 μm, at most 35 μm. In some embodiments, the first capture beads are GigaCap Q650S.

[0225] In some embodiments, the second anion chromatography capture step includes a fixation step (e.g., phosphorylation fusion polypeptide is bound to a chromatographic column), a pre-elution washing step, and an elution step (e.g., eluting the phosphorylation fusion polypeptide). In some embodiments, resin beads having a particle size of about 10 to about 50 microns (mean value), such as 35 microns (mean value) are used to fix the phosphorylation fusion polypeptide. In some embodiments, a fixed composition (e.g., using 0M sodium chloride) is used during the phosphorylation fusion polypeptide is fixed to the chromatographic column. In some embodiments, a pre-elution composition (e.g., using 274mM sodium chloride) having an intermediate salt concentration comparable to the salt concentration of the fixed composition and the elution composition is utilized during the pre-elution washing step. In some embodiments, an elution composition (e.g., using 355mM sodium chloride) is used during the phosphorylation fusion polypeptide is eluted from the chromatographic column. 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 200-400 cm / h is used (eg, 300 cm / h).

[0226] Optional additional steps

[0227] In some embodiments, the method according to the present disclosure includes a virus inactivation step or a virus removal step. In some embodiments, the virus inactivation step or the virus removal step is performed before or after any one of the chromatography steps described above. In one embodiment, the virus inactivation step or the 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., by using a chemical agent such as a surfactant). In some embodiments, the virus inactivation step includes the use of a detergent because, without being bound by a particular theory, IL12-ABP is sensitive to low pH and can aggregate at low pH. In some embodiments, the virus inactivation step includes the use of a detergent selected from myristyl dimethylamine N-oxide, TDAO, Triton X-100 (Triton X-100) or polysorbate. In some embodiments, the virus removal step includes a filtration step.

[0228] Characterization

[0229] Among other things, in some embodiments, the present disclosure provides techniques for characterizing fusion polypeptides (e.g., phosphorylated or non-phosphorylated) or preparations thereof and / or complexes comprising such fusion polypeptides and metal hydroxides, or preparations of such complexes.

[0230] In some embodiments, one or more characterizations may be performed during and / or after one or more steps of a manufacturing process (e.g., a purification process) as described herein. In some embodiments, a particular manufacturing process may be modified or terminated based on the characterization (e.g., if a particular formulation fails to meet one or more specifications). In some embodiments, characterization may involve assessing one or more of metal hydroxide retention, degree of phosphorylation, heterogeneity of phosphorylation, signaling activity, and / or efficacy. See Example 3 under "Data Analysis" for exemplary suitable characterization assays.

[0231] In some embodiments, after purifying the phosphorylated fusion polypeptide (for example, by one or more chromatographic 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 the various methods available in this area. In some embodiments, for example, but not limited to, phosphorylated fusion polypeptide is measured using malachite green (malachite green) determination, analytical ion exchange, spectrophotometer, colorimetric determination, complete mass analysis by mass spectrometry and / or protein blotting (western blot). Referring to the exemplary suitable characterization assay under "data analysis" in Example 3.

[0232] Exemplary Characterization of Phosphate Content

[0233] In some embodiments, the degree of phosphorylation is characterized (e.g., the degree of phosphorylation of a fusion polypeptide of the present disclosure). Various methods are available to measure the degree of phosphorylation (e.g., the average number of phosphate molecules per polypeptide). For example, in some embodiments, the degree of phosphorylation can be determined by colorimetry. In some embodiments, the colorimetric method is or includes a malachite green assay. Without wishing to be bound by any one theory, the malachite green assay is based on quantification (e.g., using a spectrophotometer or plate reader) of a measurable green complex formed between malachite green, molybdate, and free orthophosphate.

[0234] 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., Example 10 for exemplary degrees of phosphorylation). In some embodiments, the degree of phosphorylation (e.g., the 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.

[0235] In some embodiments, the heterogeneity of phosphorylation of a fusion polypeptide of the present disclosure and / or its formulation is characterized. In some embodiments, the heterogeneity of phosphorylation is a measure of the extent of phosphorylation within a given formulation of a fusion polypeptide. In some embodiments, the heterogeneity of phosphorylation is a measure of the extent of phosphorylation within multiple formulations of a fusion polypeptide. In some embodiments, the heterogeneity of phosphorylation is a measure of the location of a specific phosphate group on a polypeptide within a given formulation of a fusion polypeptide. In some embodiments, the heterogeneity of phosphorylation is a measure of the location of a specific phosphate group on a polypeptide within multiple formulations of a fusion polypeptide.

[0236] Various techniques are available to measure phosphorylation heterogeneity. For example, in some embodiments, the extent of phosphorylation can be determined by chromatography as described above.

[0237] In some embodiments, the differentially phosphorylated polypeptide is dephosphorylated. In some embodiments, dephosphorylation includes the use of a phosphatase (e.g., lambda phosphatase). In some embodiments, the fusion polypeptide is incubated with a phosphatase for a period of time and incubated at a temperature that allows the phosphatase activity and the dephosphorylation of the fusion polypeptide. In some embodiments, dephosphorylation occurs at about 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C or higher incubation temperatures. In some embodiments, the incubation time for dephosphorylation is 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, 65 minutes or longer. In some embodiments, dephosphorylation occurs during 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.

[0238] In some embodiments, the differentially phosphorylated polypeptides are dephosphorylated prior to isolation.In some embodiments, the differentially phosphorylated polypeptides of the present disclosure are assessed relative to an appropriate reference standard (e.g., a dephosphorylated and / or non-phosphorylated form of the fusion polypeptide).

[0239] 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, differentially phosphorylated polypeptides are measured using a malachite green assay, analytical ion exchange, spectrophotometry, colorimetric assay, and / or Western blotting.

[0240] Exemplary Characterization of Metal Hydroxide Retention

[0241] In some embodiments, the fusion polypeptides of the present disclosure form complexes with metal hydroxides (e.g., aluminum hydroxide) when exposed to the complexes. In some embodiments, the retention of the fusion polypeptides of the present disclosure on metal hydroxides (e.g., metal hydroxide retention) is characterized. Various methods are available to measure 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 optical mode spectroscopy, attenuated total internal reflection infrared spectroscopy, circular dichroism spectroscopy (CD), total internal reflection infrared spectroscopy (TIRF), and other high-resolution microscopy techniques.

[0242] In some embodiments, an in vitro assay is used to characterize metal hydroxide retention. For example, a known concentration of a 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 blotting, bicinchoninic acid assay, or Bradford assay.

[0243] 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) a metal hydroxide when mixed with it.

[0244] Exemplary characterization of signaling activity

[0245] In some embodiments, a fusion polypeptide (and / or a complex thereof) as described herein is characterized by an activity (e.g., signaling activity). In some embodiments, the activity is characterized by assessing signaling activity (e.g., signaling capacity) compared to an appropriate reference standard. An appropriate reference standard can be, for example, a wild-type polypeptide and / or a fusion polypeptide lacking a metal hydroxide binding polypeptide.

[0246] Various methods are available to assess signaling capacity. In some embodiments, for example, signaling capacity is assessed using in vitro or in vivo activity assays.

[0247] In some embodiments, signaling activity is assessed using an in vitro activity assay. In some embodiments, the in vitro activity assay includes measuring activation or inhibition of downstream signaling of the fusion polypeptide. In some embodiments, measuring activation or inhibition of downstream activity includes using a reporter gene (e.g., a reporter gene assay). In some embodiments, a reporter gene assay uses a detectable molecule (e.g., a reporter gene) associated with the fusion polypeptide activity to measure activity.

[0248] In some embodiments, reporter gene comprises fluorescence, bioluminescence and / or other detectable probes known to those skilled in the art. In some embodiments, reporter gene includes the use of gene reporter gene. For example, gene reporter gene can be activated after the signal transduction that polypeptide triggers. For example, after the activated gene reporter gene is transcribed, detectable product or enzyme can be utilized, which is activated after adding substrate to produce detectable product and / or by-product. In some embodiments, enzyme useful according to reporter gene determination is such as luciferase or alkaline phosphatase (such as secretory alkaline phosphatase, SEAP). In some such embodiments, HEK-blue-IL12 reporter gene detection is utilized.

[0249] In some embodiments, signal transduction 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, for example, by measuring activation or inhibition of downstream signal transduction of the fusion polypeptide compared to an appropriate reference standard (e.g., the activity of the wild-type polypeptide). Various methods are available to measure activation or inhibition of downstream signal transduction of the fusion polypeptide. For example, but not limited to, changes in differential gene expression, protein expression, and / or post-translational modification induced by the fusion polypeptide can be measured.

[0250] Exemplary Efficacy Characterization

[0251] In some embodiments, efficacy can be characterized according to one or more of the various methods available. In some embodiments, for example, a fusion polypeptide (or a complex thereof) as described herein is administered to a subject (e.g., a mouse, a non-human primate, a human, etc.) (e.g., by intratumoral or peritumoral injection), and efficacy is determined by comparison with an appropriate reference standard. An appropriate reference standard can be, for example, a wild-type polypeptide and / or a polypeptide lacking a metal hydroxide binding polypeptide or a polypeptide having a metal hydroxide binding polypeptide in an unbound (e.g., unphosphorylated) state.

[0252] In some embodiments, efficacy is determined preclinically in an animal model (e.g., in mice, rats, non-human primates, etc.). In some embodiments, the fusion polypeptide is administered to the animal model (e.g., by intratumoral or peri-tumoral injection). For example, in some embodiments, the animal model is an animal model with a tumor (e.g., an animal model of cancer). In some embodiments, a cancer animal model is produced by inoculating the animal model with tumor cells. In some embodiments, the animal model is inoculated with tumor cells in the lateral region. In some embodiments, the animal model is inoculated with tumor cells in a clinically relevant area (e.g., mammary fat pad).

[0253] In some embodiments, a fusion polypeptide of the present disclosure (e.g., a preparation thereof, such as a preparation of a phosphorylated fusion polypeptide) is administered to an animal model of cancer. In some embodiments, a reference standard (e.g., a wild-type polypeptide and / or a polypeptide lacking a metal hydroxide binding polypeptide) is administered to an animal model of cancer. In some embodiments, one or more of various available, predetermined efficacy measures known in the art, such as, for example, tumor volume and / or percent survival, are assessed over time relative to an appropriate reference standard (e.g., a wild-type polypeptide and / or a polypeptide lacking a metal hydroxide binding polypeptide).

[0254] In some embodiments, the efficacy of the fusion polypeptide (e.g., a preparation thereof, such as a preparation of a phosphorylated fusion polypeptide) is determined clinically. In some embodiments, the fusion polypeptide is administered to a subject with a tumor (e.g., by intratumoral, peri-tumoral injection, or administration into a tumor-draining lymph node). In some embodiments, various available, predetermined efficacy measurements known in the art, such as, for example, tumor volume and / or survival percentage, are assessed over time relative to subjects with tumors administered a reference standard (e.g., a treatment with known efficacy in the art and / or a placebo).

[0255] Formation and preparation of fusion polypeptide-metal hydroxide complexes

[0256] In some embodiments, the phosphorylated form of the fusion polypeptide described herein forms a complex with a metal hydroxide (e.g., aluminum hydroxide) when exposed thereto. In some embodiments, the fusion polypeptide comprises a hydroxyl surrogate group (e.g., a phosphate group) for adsorption via ligand exchange with the metal hydroxide. In some embodiments, the fusion polypeptide can form a complex with the metal hydroxide via electrostatic interactions.

[0257] In some embodiments, a phosphorylated form of a fusion polypeptide comprising an IL-12 immunomodulatory domain as described herein forms a complex with aluminum hydroxide upon exposure thereto, thereby forming an IL-12 complex according to the present disclosure.

[0258] In some embodiments, the fusion polypeptide metal hydroxide complex of the present disclosure (e.g., IL-12 complex) is formed by mixing. In some embodiments, mixing occurs in a buffer (e.g., tris-buffered saline buffer). In some such embodiments, the buffer does not contain phosphate. In some such embodiments, the buffer does not contain one or more substances that dissolve metal hydroxides (e.g., citric acid, malic acid, or lactic acid). Without wishing to be bound by any one theory, buffers containing phosphate compete with the formation of the complex and may hinder the formation of the complex. In some embodiments, mixing occurs at a specific temperature for a period of time. In some such embodiments, the duration is 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, or 45 minutes. In some such embodiments, the specific 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.

[0259] In some embodiments, the present disclosure provides, among other things, a fusion polypeptide-metal hydroxide complex formulation comprising a fusion polypeptide-metal hydroxide complex according to the present disclosure. In some embodiments, the formulation comprising the fusion polypeptide-metal hydroxide complex comprises any one of various suitable metal hydroxides known in the art.

[0260] In some embodiments, the present disclosure provides, among other things, a pharmaceutical composition comprising a fusion polypeptide disclosed herein. In some embodiments, the pharmaceutical composition is formulated as a fusion polypeptide-metal hydroxide complex. In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative, and / or adjuvant.

[0261] Table 1: Exemplary amino acid sequences

[0262]

[0263]

[0264]

[0265]

[0266] Table 2. Exemplary nucleic acid sequences

[0267]

[0268]

[0269]

[0270]

[0271] Example

[0272] Example 1: Optimization of production of exemplary fusion polypeptides

[0273] This example demonstrates that stable pools of cells transfected with a leap-in transposase construct comprising nucleotides encoding an IL-12 fusion polypeptide and nucleotides encoding hFAM20C can be established and used to produce IL-12 fusion polypeptide. The example also demonstrates that stable pools of transfected cells are capable of producing large amounts of IL-12 fusion polypeptide.

[0274] Leap-in transposase

[0275] The transposon system used in this embodiment is designed to efficiently and continuously deliver IL-12 fusion polypeptide and hFAM20C to host cells (e.g., CHO cells). The transposon system includes a transposon construct (DNA) and a homologous transposon enzyme (mRNA) (also referred to as leap in enzyme or transposase). The transposon construct is produced by synthesizing the nucleotide sequences encoding IL-12 fusion polypeptide and hFAM20C and inserting them into a double open reading frame (ORF) expression construct. The transposon construct is also designed to express and deliver glutamine synthase (GS).

[0276] Transposases belong to the DDE / D integrase family and catalyze an efficient cut-and-paste (excision followed by integration) process to integrate their cognate transposons into the target genome. The enzyme's mechanism of action results in the generation of single-copy integrons in multiple genomic loci. This homologous integration mechanism is several orders of magnitude more efficient than nonhomologous recombination. Furthermore, it eliminates concatemer formation and deleted and rearranged transgenes. Leap-In-mediated transposition enriches integration sites in transcriptionally active genomic regions.

[0277] One of the striking features of the Leap-In transposase-mediated stable integration mechanism is the strong comparability between Leap-In-mediated stable pools and monoclonal cell lines derived from those pools. This comparability applies to both productivity and global basic physicochemical properties.

[0278] In order to ensure that the integration event is irreversible so that the transgene can be stably integrated, the transposase is delivered as mRNA. About 2 days after the cells were transfected with mRNA, no enzyme was detected, and therefore it is believed that no further transgene excision / reintegration can occur. Internal (In-house) long-term (> 3 months) experiments showed that there was no natural internal enzyme activity of the transgenic gene that could be moved through the transposition integration in the host CHO cells. Finally, no integrase (transposase) was detected in the master cell bank established by using the clinical manufacturing cell line developed by the Leap-In system.

[0279] The transposase mRNA manufacturing process utilizes recombinant protein components but contains no animal-derived raw materials.

[0280] CHO cell lines

[0281] A Chinese hamster ovary (CHO) cell line null for glutamine synthase (GS) was developed (GS-CHO cell line) and used as the parental cell line (producer cell line). The glutamine synthase (GS) gene was knocked out, rendering the resulting cells unable to synthesize their own glutamine and completely dependent on glutamine supplementation for growth. The presence of GS on the transposon construct used for transfection allows selection of successful transfectants in glutamine-free culture medium.

[0282] IL-12 fusion protein:Fam20C ratio study

[0283] Experiments were performed to establish a ratio of IL-12 fusion protein:Fam20C that achieved optimal cell growth and high productivity of the fusion polypeptide.

[0284] Stable pool transfections were generated at 2:1, 4:1, and 8:1 ratios. See Table 3.

[0285] Table 3: 2:1, 4:1, and 8:1 ratios

[0286] Pool Protein name Production Run ID 5629P1 4:1D8NoHis 1:D7 5630P2.1 2:1D8NoHis 1:D7 8702 5630P2.2 2:1D8NoHis 1:D7 (repeat) 8734 5631P3 8:1D8NoHis 1:D7 8823 5632P4 4:1D8NoHis 1:D22 8824

[0287] Endpoint viability was >90% for all pools except 5630P2.2 (replicate) which dropped to 80% (see Figure 3A Peak viable cell density (VCD) ranged from 18 to 27e6 / mL ( Figure 3B ). The lowest viability was observed for 5630P2 (2:1 ratio). The highest viability was observed for 5631P3 (8:1) and 5631P4 (4:1). Overall, 5631P3 showed the best cell growth based on a combination of viability and visible cell density (VCD). Some cell lines can achieve higher VCDs but die faster, which is not ideal.

[0288] The production of cellular IL-12 fusion polypeptide was measured by IL-12 Octet assay. The results are shown in Table 4.

[0289] Table 4 Productivity

[0290]

[0291] The highest productivity was observed for 5631P3 (8:1 ratio). When SDS-PAGE analysis was performed, a clear product band was observed for 5631P3 (production run number 8823). Figure 3C ).

[0292] In addition to the 2:1, 4:1 and 8:1 transfections, six pools were generated at ratios of 15:1 and 8:1, see Table 5.

[0293] Table 5: Six cell pools were generated by transfecting stable double OFR constructs into GD-CHO cells.

[0294]

[0295] Viability and viable cell density (VCD) are shown in Figure 4A and Figure 4B All six cell pools showed viability >85% and peak VCD ranged from 15-23e6 / mL. Both constructs showed good cell growth.

[0296] Productivity was measured by IL12 Octet assay and is shown in Figure 5 The stable cell pools produced 50 mg / L to approximately 900 mg / L of the fusion polypeptide. Pool 11370 (D208_8:1) exhibited higher productivity than pool 11373 (D208_15:1). This demonstrates that cell lines transfected with an 8:1 (IL-12 fusion polypeptide:Fam20C kinase) ratio provide stable cell lines with good viability and high IL-12 fusion polypeptide production.

[0297] Example 2: Generation of a stable pool based on an 8:1 ratio

[0298] This example supports that a pool of stably transfected cells expressing a fusion polypeptide and a kinase at a ratio of approximately 8: 1 is capable of producing large amounts of IL-12 fusion polypeptide. Furthermore, these cells are capable of producing a phosphorylated form of the fusion polypeptide.

[0299] In Example 2, the GS-CHO cells and Leap-In transposon system described in Example 1 were used.

[0300] 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.

[0301] Transfection

[0302] Untransfected GS-CHO cells were maintained in shake flasks with growth medium and kept in a shaker incubator at 5% CO2, 37°C and 70-80% relative humidity.

[0303] 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 to 48 hours in growth medium in a T25 flask under static conditions. After the recovery period, the transfectant culture was introduced into metabolic selection by exchanging the cell culture medium for a glutamine-free formulation.

[0304] Fed-batch production and fed-batch operation

[0305] Fed-batch production runs were performed in a TubeSpin bioreactor for pool ranking. The working volume was 10 mL and the shaker speed was 240 rpm.

[0306] On day 0 of a 14-day fed-batch run, cells were seeded in a production medium containing 4 mM glutamine at a density of 0.75 x 106 cells / mL. After day 0, no additional glutamine was added to the fed-batch culture. The culture was maintained at 37°C, 5% CO2, and 70-80% relative humidity. When the density of the culture reached 10-15 x 106 cells / mL, the culture temperature was reduced to 32°C for the remainder of the run duration. If viability dropped to 75%, the production run was harvested on day 14 or earlier. Cell counts and metabolite measurements were performed on days 4, 7, 10, 12, and 14. On the feed day, up to 12 g / L of glucose (depending on the consumption rate) was added to the culture using a sterile 45% glucose solution. The pH was not adjusted during the pilot production run.

[0307] Harvest and culture clarification

[0308] Harvested material from production cultures was clarified by centrifugation (3000 g, 20-30 min) and then filtered through a 0.22 μm membrane.

[0309] analyze

[0310] SDA Page method: For reducing SDS-PAGE gel, in NuPAGE TM The clarified supernatant was prepared in LDS sample buffer (4X) (ThermoFisher Cat. No. NP0008) and analyzed using NuPAGE according to the supplier's protocol. TM Sample reducing agent (10X) (Cat. No. NP0009) was used for reduction. According to the supplier's protocol, the reduced samples were run on NuPAGE Novex 4-12 Bis-Tris protein gels (Cat. No. NP0329BOX, WG1403BX10) and the InstantBlue TM Protein stain (Novus Biologicals catalog number ISB1L-1L) was used for staining. For non-reducing SDS-PAGE gels, the NuPAGE TM Clarified supernatants were prepared in LDS sample buffer (4X) (Cat. No. NP0008). 10 mM N-ethylmaleimide (NEM) (MilliporeSigma Cat. No. E3876-5G) was added to the samples to prevent disulfide bonds from reforming. Samples were run on NuPAGE Novex 4-12 Bis-Tris protein gels (Cat. No. NP0329BOX, WG1403BX10) according to the supplier's protocol and analyzed using InstantBlue™. TM Protein stain (Cat. No. ISB1L-1L) was used for staining.

[0311] Results and discussion

[0312] Pool Vitality

[0313] GS-CHO cells were transfected with expression constructs expressing IL-12 fusion polypeptide and Fam20C kinase to generate a pool of transfected cells (6880P1) that produced IL-12 fusion polypeptide and hFAM20C that phosphorylated IL-12 fusion polypeptide. An 8:1 ratio of IL-12 fusion polypeptide:Fam20C was used. After a 2-day recovery period after transfection, cells were seeded into selection medium at 0.3x106 cells / mL. This selection pressure was maintained throughout the selection period. During the selection period, viable cell density and percent viability were determined 2-3 times per week. The viability during selection is shown in Figure 2. Figure 6 middle. Figure 6 A general decrease in cell viability was shown after transfection, but importantly, the cell line showed good recovery and long-term high viability (e.g., from about day 18 to day 43 in culture). In summary, healthy and stable cells were generated that appeared to be transfected with a construct having an 8:1 ratio of IL-12 fusion polypeptide:Fam20C.

[0314] Cell productivity and fusion peptide quality

[0315] In TubeSpins, the fed batch production operation of 10mL cultivation is started for cell pool 6880P1 to estimate productivity and protein quality. The productivity in the fed batch process is measured by biolayer interferometry (BLI) method. In addition, SDS-PAGE is also used to assess productivity and product quality. The harvest of clarification is diluted with sample diluent (Pall ForteBio catalog number (Cat. No.) 18-1048), and measured by biolayer interferometry (BLI) in 96 orifice plates on Octet HTX instruments using protein A sensor. Suitable sample dilutions are produced, and all valid measured values are within the standard concentration range. Various repeated measurements have also been carried out to reduce the measurement error associated with dilution and sensor. IL12 samples are measured on OctetHTX instruments (ForteBio) using standard protocols. Mouse anti-human IL12 antibody (BioLegend catalog number (Cat. No.) 508808) is loaded onto anti-mouse Fc and captured on tip (AMC, ForteBio). The IL12 fusion polypeptide concentration was calculated in ForteBio DataAnalysis 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) between 1.56 and 100 μg / ml. The clarified supernatant was diluted into 1X Kinetics buffer (ForteBio) so that the measured concentration was within the standard curve range. Additional qualitative analysis was performed by SDS-PAGE. The performance parameters of Pool 6880P1 during the fed-batch production evaluation are shown in Figure 7A -C.

[0316] Transfected cells showed viability greater than 90% during the 14-day fed-batch run ( Figure 7A ), and they were able to produce 100 mg / L (day 7) to 150 mg / L (day 14) of IL-12 fusion polypeptide ( Figure 7C ).

[0317] Malachite green assay was performed on purified IL-12 fusion polypeptide from pool 11370, harvested on day 14. The IL-12 fusion polypeptide was purified using a first anion chromatography step and a hydrophobic interaction chromatography step. This purification step did not include a second anion exchange polishing step. The IL-12 fusion polypeptide exhibited approximately five phosphates per fusion polypeptide.

[0318] Example 3: Bioreactor Production of IL-12 Fusion Polypeptides

[0319] The transfected GS-CHO cells from Example 2 were further cultured and tested.

[0320] Using a fluorescence-activated cell sorter, only GS-CHO cells expressing detectable levels of IL-12 fusion polypeptide were sorted into 96-well plates at a single cell per well. A total of approximately 5,700 wells were targeted for single cell seeding.

[0321] 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 top-ranked cell lines were transferred to suspension culture.

[0322] After transfer to suspension culture, the microbioreactor system ( The growth and productivity of 24 top-ranked cell lines were evaluated using a TAPBiosystems 250 system (Sartorius Stedim Biotech). The 24 cell lines were selected based on IL-12 fusion polypeptide product concentration, as assessed by static productivity assessment, and acceptable cell line growth (viable cell concentration (VCC) consistently above 1.0 x 106 cells / mL and culture viability above 90% on the day of subculture). Twenty-three cell lines were successfully evaluated. IL-12 fusion polypeptide concentrations at harvest, as determined by Octet analysis, ranged from 189.1 to 742.8 mg / L.

[0323] Based on the high productivity (for example, at least 180mg / mL) in the fed-batch micro-bioreactor assessment and the acceptable growth characteristics (for example, always higher than 1.0x 10 in the subculture Individual viable cells / mL) during conventional subculture in shake flask culture, eight cell lines were selected for further evaluation. The images of the screening phase were also examined to support that the main candidate cell line emerged from a single bacterium colony (FACS was operated in single cell sedimentation mode). Growth and productivity data from the FMB1 (fed-batch culture) of 8 selected main candidate cell lines are presented in Table 6, as well as the lactic acid concentration during the corresponding harvest date, results and the osmotic pressure during the results.

[0324] Table 6: Exemplary host cells expressing IL-12 fusion proteins in fed-batch microbioreactor evaluation.

[0325]

[0326] A 12-vial research cell bank of each of the eight selected lead candidate cell lines was cryopreserved.

[0327] use The system was purified by CH1 affinity purification of the product of the harvested clarified conditioned medium (CCS) from FMB1 cultures of eight lead candidate cell lines (Section 4.17). The partially purified supernatant was characterized by N-linked oligosaccharide analysis using UPLC-MS, aggregate / fragment analysis using GP HPLC, N-linked and O-linked oligosaccharide analysis using PMAP-MS, intact protein structure characterization using ESI-MS, phosphorylation analysis using malachite green, sialic acid determination using RP HPLC, and purity determination using RP UPLC.

[0328] Data Analysis

[0329] use System for protein concentration determination

[0330] The HTX system (FortéBio Biologics / Molecular Devices LLC) is used to perform biolayer interferometry on CCS samples to enable ranking by product concentration. The system uses a glass fiber biosensor coated with a special optical layer, and the capture molecules are attached to the tip. The tip is immersed in a sample containing the target molecule. The target molecule binds to the capture molecule, and the two form a molecular layer. When white light enters the biosensor, two beams of light are reflected to the back end: 1. from the tip as a reference, 2. from the molecular layer. The interference of the two beams of light causes a wavelength shift that varies with the thickness of the molecular layer and corresponds to the number of molecules on the surface of the tip. When the target molecule binds to the biosensor, the wavelength change expressed in nanometers is plotted in real time to generate a binding curve. Concentration-dependent binding curves are obtained for standards, controls, and samples, which represent the rate of increase in the thickness of the molecular layer when the product binds to the capture molecules on the surface of the biosensor tip under constant conditions. Use The data analysis software determines the on-rate from the binding curve. The on-rates of standards of known concentration are plotted into a standard curve, which is used to estimate the product concentration in the sample and thus rank the product concentration.

[0331] use Hydroxyapatite type 1 resin (CH1) affinity purification

[0332] For analytical purposes only, product purification is performed using CaptureSelect CH1-XL according to preset binding and elution conditions. When the solution containing the product is passed through the resin, the molecule of interest reversibly binds to the CH1 affinity ligand, allowing impurities to flow through the column. The bound product is then recovered by lowering the pH with elution buffer, which denatures the resin-product bond. Thus, the product can be separated from contaminants. After purification, the sample is neutralized and prepared for product quality analysis. The concentration of the eluate is measured using the Nanodrop system.

[0333] UPLC-MS analysis

[0334] The UPLC-MS high-throughput N-glycan analysis platform includes high-throughput glycan preparation using the GlykoPrep Rapid2-AB kit and the AssayMAP Bravo liquid handler, followed by UPLC and mass spectrometry analysis. The sample preparation workflow consists of automated purification and normalization steps, followed by release of N-glycans by digestion with the enzyme peptide-N-glycosidase F, 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 online using an AQUITY UPLC H-Class Bio System with an AQUITY UPLC fluorescence detector and a tandem Xevo G2S Q-TOF system, operated in sensitivity mode and positive ionization mode.

[0335] Oligosaccharide structures were quantified and identified using the glycan workflow in UNIFI 1.8.2 software. A 2-AB-labeled dextran ladder was used to calibrate and normalize the retention times of 2-AB-labeled glycans to glucose units. Tentative assignments of oligosaccharide species were made for both neutral and charged oligosaccharide analyses based on comparison of glucose units with the NIBRT glycan database. These tentative assignments were confirmed by mass analysis. The percentage of each glycan was based on the area of each peak relative to the total integrated peak area. Using this method, some less abundant peaks could not be identified because their proportions were too low to be detected by mass spectrometry, yet they had glucose unit values similar to entries in the NIBRT glycan database. For this reason, peaks with concentrations below 0.3% were not reported.

[0336] Gel permeation (GP) HPLC analysis

[0337] GP HPLC was used to separate the product monomers from aggregates and fragments. Monomeric components were identified by their characteristic retention times and positions relative to calibration markers. Aggregate analysis was performed using a G3000SWXL column (Hichrom Ltd). Product components were detected by A280 nm measurement and analyzed using Empower TM Peak chromatograms were analyzed using ELISA® 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 area used to measure the product concentration in the sample.

[0338] Peptide mapping mass spectrometry (PMAP-MS) analysis

[0339] PMAP-MS analysis was performed using a Waters Acquity UPLC and a Waters XevoG2 QTO. Samples were trypsin digested. Prior to analysis, the MS was calibrated for mass assignment, resolution, and sensitivity. A Glu-fibrinogen lockspray was acquired every 30 seconds as a reference scan. Data analysis was performed using MassLynx and BiopharmaLynx software. This PMAP-MS analysis focused on comparing peaks within the total ion chromatogram (TIC). MS1 and MS / MS data were acquired for all samples. Only MS1 data were used for analysis; the MS / MS dataset provided additional information where needed. The following were not covered in this analysis: sequence coverage estimation, clipping, pyroglutamination, deamidation, glycation, and oxidation.

[0340] Electrospray ionization mass spectrometry (ESI-MS) analysis

[0341] ESI-MS analysis was performed using a Waters Acquity UPLC and a Waters Xevo G2 QTOF. Due to sample limitations (cloning buffer components were compatible with this method), samples were deglycosylated and reduced according to UKSL-1795 without buffer exchange. LCMS analysis was performed using a Xevo G2 QTOF mass spectrometer operating in positive ion mode from 600 m / z to 4000 m / z. Data analysis was performed using Protein Metrics software (version 3.9) utilizing a minimalistic deconvolution algorithm. Appropriate processing parameters were selected for the product being analyzed, and the masses assigned during this processing are reported.

[0342] Malachite green

[0343] Using Thermo Scientific TMThe Phosphoprotein Phosphate Estimation Assay Kit determines the degree of phosphorylation of purified IL-12 fusion polypeptides. The kit utilizes alkaline hydrolysis of phosphate groups from seryl and threonyl residues in phosphoproteins. The released phosphate forms a complex with added ammonium molybdate. Malachite green reagent then forms a secondary complex with phosphomolybdate, producing a color change reaction. The sample's absorbance is measured using a microplate reader and estimated based on a 5-point standard curve of known phosphate concentrations.

[0344] Sialic acid determination

[0345] Sialic acid determination in the test samples was performed by reversed-phase (RP) HPLC. The sample preparation workflow consisted of diluting the test sample and then hydrolyzing it with a TFA solution. The hydrolyzed test sample was then labeled with a DMB solution to form a fluorescent reaction product. RP-HPLC with fluorescence detection was used to quantify N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) based on external standards. Samples were analyzed by reversed-phase (RP) HPLC using an Agilent 1100 / 1200 series HPLC system.

[0346] Reversed-phase ultra-performance liquid chromatography (RP-UPLC) analysis

[0347] RP-UPLC with fluorescence detection (FLD) is used to quantify the relative percentage of the total purity of the IL-12 fusion polypeptide product. Reversed-phase chromatography separates sample components based on differences in hydrophobicity. The sample is injected onto the column (stationary phase), and interactions with the hydrophobic surface remove them from the flowing mobile phase. Sample components bind to the column depending on the strength and nature of the hydrophobic and polar interactions. The product is eluted by increasing the organic strength of the mobile phase. Components with lower hydrophobicity elute earlier, and components with higher hydrophobicity elute later. Eluted components are monitored using fluorescence.

[0348] result

[0349] The IL-12 fusion polypeptides produced by all eight cell lines (NKB05, NKB19, NKB23, NKB28, NKB44, NKB46, NKB70, NKB78) were similar when analyzed for aggregates by GP HPLC, for phosphorylation by malachite green, and for sialic acid by RP-HPLC. Some differences were observed in the proteins produced by these cell lines when analyzed for oligosaccharides by UPLC-MS and for major phosphorylation sites by PMAP-MS, but these differences were not sufficient to exclude any of the cell lines from further evaluation.

[0350] Table 7: PMAP-MS analysis of phosphorylation in AKTA-purified ANK-101 from FMB1 cultures of eight lead candidate cell lines.

[0351]

[0352]

[0353] Finally, differences in intact protein characterization data were observed in 2 of the cell lines, indicating the presence of product fragments in these cell lines.

[0354] Characterization data of major cell lines

[0355] Table 8: Summary of growth and productivity data for eight selected cell lines expressing IL-12 fusion polypeptides in FMB1.

[0356]

[0357]

[0358] Table 9: Summary of malachite green analysis of AKTA-purified IL-12 fusion polypeptides from FMB1 cultures of eight lead candidate cell lines.

[0359] Cell line ID Number of phosphorus molecules per protein molecule NKB05 8.7 NKB19 7.8 NKB23 8.3 NKB28 8.3 NKB44 6.5 NKB46 7.7 NKB70 9.2 NKB78 6.9

[0360] Table 10: Summary of sialic acid data for AKTA-purified IL-12 fusion polypeptides from FMB1 cultures of eight lead candidate cell lines.

[0361]

[0362] Following evaluation in fed-batch microbioreactors, cell line NKB46 was selected as the interim master cell line for preparation of the master cell bank and future cGMP production of IL-12 fusion peptides. This selection was based on high productivity, high specific production rate, and acceptable growth in fed-batch microbioreactor evaluations, as well as product characteristics (including phosphorylation levels) in fed-batch microbioreactor evaluations. For current Good Manufacturing Practice (cGMP) purposes, this cell line was renamed INTRA2021.

[0363] Example 4: Exemplary Purification Overview

[0364] This example shows an exemplary method for purifying IL-12 fusion polypeptides from transfected cells.

[0365] Exemplary purification methods include:

[0366] • Harvest the bioreactor (typically 2 weeks, e.g. for fed-batch runs as in Examples 2 or 3) by depth filtration or centrifugation to remove cells and cell debris.

[0367] For CHO-based processes, inactivate viruses by low pH or detergents.

[0368] A first chromatography step, such as a capture step (e.g., an anion exchange capture chromatography step)

[0369] A second chromatography step, such as a polishing step (e.g., hydrophobic interaction chromatography)

[0370] A third chromatography step, such as a final polishing step (e.g., an anion exchange capture chromatography step)

[0371] UF / DF for formulation of bulk drugs in the correct buffers and excipients

[0372] Example 6: Conventional capture chromatography

[0373] Equilibrate the resin / column with an appropriate buffer and adjust the column to the correct pH (e.g., 6.8-7.4) and conductivity (salt, such as NaCl). Load the column with the appropriate load (clarified cell culture medium, which typically matches the pH and conductivity of the equilibration buffer). Wash the load with the equilibration buffer until all load material is removed from the column (A280 drops to baseline). Perform another wash to remove impurities before eluting the product (IL-12 fusion polypeptide). Elute the phosphorylated fusion polypeptide by increasing / decreasing the salt concentration or changing the pH. Strip any remaining bound material from the column using high / low salt, chaotropes, alcohol, or NaOH.

[0374] Example 7: Purification-1

[0375] This example shows 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 the fusion polypeptide from lower and higher molecular weight impurities (e.g., aggregated IL-12 fusion polypeptide) and host cell impurities, and that it is possible to obtain a highly pure preparation of the phosphorylated form of the fusion polypeptide.

[0376] In this example, a first anion chromatography (AEX) step was used to capture the IL-12 fusion polypeptide from a cell extract. 1) a hydroxyapatite chromatography capture step or 2) a hydrophobic interaction polishing chromatography step was then used as a subsequent polishing step.

[0377] Culture harvest and clarification

[0378] Fed batch harvesting ended at the 14th day and was clarified by centrifugation. Stored frozen in 200 mL aliquots. The aliquots were thawed in a room temperature bath and clarified by secondary depth filtration. Use DI H o rinse filtrate and perform feasibility testing after balancing with TBS (20 mM Tris pH 7.4, 150 mM NaCl).

[0379] Anion chromatography capture step (AEX)

[0380] An exemplary method for anion exchange capture chromatography is shown below.

[0381] Column: Tosoh GigaCap Q-650M, 5 mL, 1.46 cm D x 3 cm H

[0382] Equilibration buffer (A): 20 mM Tris pH 7.4

[0383] Elution buffer (B): 20 mM Tris pH 7.4 + 1 M NaCl

[0384] Wash buffer: 0.5N NaOH

[0385] Load preparation: The clarified harvest was diluted 1:1 (volume:volume) with DI H2O.

[0386] The eluted fractions were stored at 4°C overnight.

[0387] Table 11

[0388]

[0389]

[0390] Figure 8 Shown is the complete chromatogram and a zoom of the elution peaks during the first chromatography analysis of the clarified harvest of the stable pool 6880P1 by anion exchange chromatography (GigaCap Q). The SDS-PAGE gel shows that the IL-12 fusion polypeptide (band approximately 75,000 Daltons) is captured by the resin and that anion exchange capture chromatography can be used to separate the IL-12 fusion polypeptide from lower and higher molecular weight impurities ( Figure 9 ).

[0391] Hydroxyapatite chromatography capture step

[0392] The IL-12 fusion polypeptide was first captured by AEX Gigacap Q (5 mL) and then purified (polished) by a polishing hydroxyapatite capture chromatography step. An exemplary method for hydroxyapatite capture chromatography is included below.

[0393] Column: BioRad CHT XT, 1 mL, 0.8 cm D x 2 cm H

[0394] EQ buffer (A): 5mM NaPO4 pH 6.8, 20ppm Ca++, 50mM NaCl, 50mM MES

[0395] Elution buffer (B): 500 mM NaPO4 pH 6.8, 20 ppm Ca++, 50 mM NaCl, 50 mM MES

[0396] Regeneration buffer: 400 mM NaPO4 pH 7.0

[0397] Wash buffer: 1N NaOH

[0398] Load preparation: Dilute XT pool 1:2 (v:v) with EQ buffer. Load 0.5x volume each time (perform 2 column runs).

[0399] Table 12

[0400] step describe volume flow rate disinfect Cleaning buffer 5CV 0.5mL / min Neutralization Regeneration buffer 10CV 1mL / min balance Equilibration buffer 5CV 1mL / min load Adjust load 25 CV 0.5mL / min washing Equilibration buffer 25 CV 0.5mL / min Elution 1 Linear: 0-50% B 20CV 1mL / min Collect fractions Elution 2 Step 50-100% B 10CV 1mL / min peeling Cleaning buffer 5CV 1mL / min

[0401] Figure 10 Shown is the complete chromatogram and a zoom of the elution peaks during the first chromatography analysis of the clarified harvest of the stable pool 6880P1 by hydroxyapatite chromatography as a second chromatographic step. The SDS-PAGE gel shows 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 eluted in fraction A, while fraction B contained significant amounts of lower molecular weight impurities.

[0402] Hydrophobic interaction polishing chromatography step

[0403] The IL-12 fusion polypeptide was first captured by AEX Gigacap Q (5 mL) and then purified (polished) by heparin HP or phenyl HP chromatography.

[0404] Table 13

[0405]

[0406]

[0407] Non-reducing SDS-PAGE was loaded at 3 μg / lane ( Figure 12 An example of comparison of phenyl chromatography (hydrophobic interaction) with hydroxyapatite (CHT) after primary capture using AEX chromatography is shown in Figure 12 middle.

[0408] The phenyl fractions (lanes 8-10) eluted with 1 M salt (NH4SO4) (indicated by circles) showed higher IL-12 fusion polypeptide purity when compared with the CHT eluted fractions (lanes 3-4). This shows that IL-12 fusion proteins with high purity can be isolated.

[0409] An exemplary method for Phenyl HP chromatography is included below.

[0410] Column: Phenyl Sepharose HP (GE 17-1082-03) 5 mL, 1 cm D x 6.4 cm H

[0411] EQ buffer (A): 20 mM Tris pH 7.4, 1.5 M ammonium sulfate

[0412] Elution buffer (B): 20 mM Tris pH 7.4

[0413] Wash 2 buffer: EQ buffer + 5% isopropanol

[0414] Wash buffer: 0.01N NaOH

[0415] Load preparation: add 7.5 mL of 4M 1.5M ammonium sulfate clear solution by centrifugation

[0416] The eluted fractions were stored at 4°C overnight.

[0417] Table 14

[0418]

[0419]

[0420] Analysis of fractions eluted from the Phenyl HP column demonstrated that the IL12 fusion molecule was of high purity as determined by SDS-PAGE and SEC-HPLC (Figure 13).

[0421] Another example of phenyl chromatography is where more elaborate loading and elution conditions yield high purity material (phenyl chromatography after an AEX capture step).

[0422] Example 8: Purification-2

[0423] This example demonstrates that phenyl chromatography reduces impurities and that Q chromatography can separate product from aggregated products and isolate specific IL-12 fusion polypeptide phosphorylation levels.

[0424] Cells were harvested and clarified as in Example 7.

[0425] Affinity chromatography capture step

[0426] An exemplary method of affinity capture chromatography is shown below.

[0427] Column: Cytiva Heparin Sepharose 6FF, 45 mL (2.6 cm D x 8.5 cm H)

[0428] Equilibration buffer (A): 20 mM Tris pH 7.4

[0429] Elution buffer (B): 20 mM Tris pH 7.4 + 2 M NaCl

[0430] Wash buffer: 0.1N NaOH

[0431] Load preparation: The clarified harvest was diluted 1:1 (volume:volume) with equilibration buffer.

[0432] Table 15

[0433]

[0434]

[0435] Example of affinity capture chromatography on heparin agarose, where the elution conditions demonstrate separation between the IL12 fusion polypeptide and lower molecular weight impurities as confirmed by SDS-PAGE analysis ( Figure 14 ).

[0436] Hydrophobic interaction polishing chromatography step

[0437] An exemplary method for Phenyl HP chromatography is included below.

[0438] Column: Phenyl Sepharose HP (GE 17-1082-03) 11 mL, 1.0 cm D x 14 cm H EQ Buffer (A): 20 mM Tris pH 7.4, 100 mM NaCl, 1.5 M ammonium sulfate Elution Buffer (B): 20 mM Tris pH 7.4

[0439] Wash buffer: 0.01N NaOH

[0440] Load preparation: Adjust the heparin elution pool to 1.5 M using 4.0 M ammonium sulfate stock solution.

[0441] Ammonium sulfate. Allow to wait 30 min and filter the load using a 0.45 micron filter (vacuum flask).

[0442] Load 0.5x volume per run (two runs total)

[0443] Table 16

[0444]

[0445]

[0446] Figure 14 Shown is a non-reducing, non-stained 4-10% SDS-PAGE gel loaded with heparin or phenyl fractions (3 μg sample per lane). The eluted fraction (lane 7) shows significant purification when compared to lane 9 (load for phenyl chromatography). In addition, SEC-HPLC analysis ( Figure 16A ) showed that the phenyl pool was purer than its load (heparin agarose eluate).

[0447] Anion chromatography capture step

[0448] An exemplary method for anion exchange capture chromatography is shown below.

[0449] Column: Cytiva Q Agarose HP, 11 mL, 1.0 cm D x 14 cm H

[0450] Equilibration buffer (A): 20 mM Tris pH 7.4

[0451] Elution buffer (B): 20 mM Tris pH 7.4 + 1 M NaCl

[0452] Wash buffer: 0.5N NaOH

[0453] Load preparation: Pool the phenyl 0.75M E1 eluates and dilute to 0.1x in buffer A.

[0454] The conductivity of the load was about 16 mS / cm.

[0455] Table 17

[0456]

[0457] Figure 15 A non-reducing, stain-free 4-10% SDS-PAGE gel loaded with 3 μg of sample per lane is shown. AEX as a final polishing step was shown to produce highly pure material (SDS-PAGE and SEC-HPLC, Figure 15 and Figure 16A ) and separation of monomeric products from aggregates (fractions 419 and 424, Figure 16B The malachite green assays of the Q HP chromatographic fractions are shown in Table 14.

[0458] Table 18

[0459]

[0460] This exemplary purification method shows that phenyl chromatography significantly reduces product and processing impurities as demonstrated by SDS-PAGE.SEC-HPLC and Q chromatography are powerful tools for separating the product from aggregated products (SEC-HPLC), other impurities, and selected phosphorylation levels (malachite green data).

[0461] Example 9: Purification-3

[0462] 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-reduced and reduced RP HPLC, respectively) with acceptably low levels of host cell proteins (HCP), DNA, low molecular weight species (LMWS), high molecular weight species (HMWS), and detergent levels. The method provides an IL-12 fusion polypeptide with the desired phosphorylation level, exhibiting approximately 7-8 phosphorus molecules per molecule of protein as determined by malachite green.

[0463] As described in previous examples herein, a laboratory scale run (LSRT) was performed on the purification process of the product IL-12 fusion polypeptide from a pool of transfected GS-CHO cells.

[0464] An exemplary purification process for ANK-101 (IL-12 fusion polypeptide) includes three chromatographic steps;

[0465] TOYOPEARL GigaCap Q-650M anion exchange chromatograph (equivalent to the Q chromatograph used in Examples 7 and 8),

[0466] Capto Phenyl HS hydrophobic interaction chromatography (equivalent to the phenyl chromatography as used in Examples 7 and 8), and

[0467] • TOYOPEARL GigaCap Q-650S anion exchange chromatograph (equivalent to the Q chromatograph used in Examples 7 and 8).

[0468] The process also includes a viral inactivation step before loading onto the first chromatography step. The second chromatography step is followed by an intermediate ultrafiltration / diafiltration (UF / DF). The third step is followed by a viral size reduction filtration and final dilution. These are all steps in the manufacturing process that can accompany the chromatography step.

[0469] Detailed purification workflow:

[0470] TDAO virus inactivation

[0471] TOYOPEARL GigaCap Q-650M anion exchange chromatography (AEX)

[0472] Capto Phenyl HS Hydrophobic Interaction Chromatography (HIC)

[0473] Intermediate ultrafiltration / diafiltration (UF / DF)

[0474] TOYOPEARL GigaCap Q-650S anion exchange chromatography (AEX)

[0475] Planova BioEX Virus Reduction Filtration (VRF)

[0476] UF / DF and excipient addition

[0477] The TOYOPEARL GigaCap Q-650M chromatography step was performed at a linear flow rate of 300 cm / h using a loading capacity of 21.4 g / L resin determined during this phase. The average step recovery based on the octet data was 100%, and the average step recovery based on the RP HPLC data was 85.7%.

[0478] The Capto Phenyl HS chromatography step was performed at a loading 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%.

[0479] Further purification experiments were evaluated on ceramic hydroxyapatite (CHT XT) and a TOYOPEARL GigaCap Q-650S. Data obtained on the TOYOPEARL GigaCap Q-650S demonstrated that the isocratic elution step achieved the desired product quality, with low impurity levels and efficient separation of phosphorylated species. A three-chromatographic process was designed for the purification of the IL-12 fusion polypeptide. The average step recovery was 51.3%.

[0480] Planova BioEX filter size results show that with a 1.0m2 membrane, the material was processed within 5 hours on a GMP scale. In some methods, the maximum volumetric flux within the 5h processing time was 262.6L / m2. The final product was diluted into 20mM tris, 50mM sodium chloride (pH 7.3) with a protein concentration of 2.040g / L. This purification step successfully recovered 1326.17mg (83.8%). Excipients were added to the product after diafiltration to achieve a final composition of 20mM tris, 50mM sodium chloride, 10mM methionine, 150mM sucrose, 0.02% polysorbate (pH 7.3) with a protein concentration of 2.006g / L. The estimated cumulative processing yield was 19.7%. The estimated cumulative processing yield was 19.7%.

[0481] Analytical testing of the bulk purified IL-12 fusion polypeptide product resulting from the final purification process showed product purity greater than 98% as measured by GP HPLC analysis, and greater than 82% and 55% as measured by non-reducing and reducing RP HPLC, respectively.

[0482] 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 product contained from about 7 to about 9 (e.g., 7) phospho-molecules, as identified by malachite green assay. Intact LC ESI-MS revealed the presence of 6X, 7X, 8X, 9X, 10X, 11X, and 12X phosphorylation sites in the bulk purified IL-12 fusion polypeptide product. Intact LC ESI-MS also revealed +9 phosphate as the most predominant species in the bulk purified product.

[0483] TOYOPEARL GigaCap Q-650M anion exchange chromatography as the first step

[0484] Below are a number of exemplary methods showing 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 and higher purity of host cell proteins (HCPs). Anion exchange chromatography also enriches for highly phosphorylated species.

[0485] An exemplary method for anion exchange capture chromatography using a linear gradient elution method is described in Table 19.

[0486] Table 19: TOYOPEARL GigaCap Q-650M chromatographic gradient elution operating conditions.

[0487]

[0488] Table 20: TOYOPEARL GigaCap Q-650M Gradient Elution Analysis - Simulation Pool Summary

[0489]

[0490]

[0491] Based on the pooling strategy, the first capture step (anion exchange chromatography) achieved a significant reduction of host cell proteins (HCPs), simulated pool 4, and higher purity.

[0492] Exemplary methods for anion exchange capture chromatography using either stepwise or isocratic conditions are described in the table below.

[0493] Table 21: TOYOPEARL GigaCap Q-650M chromatography isocratic elution operating conditions.

[0494]

[0495] An exemplary method for anion exchange capture chromatography is described in the table below.

[0496] Table 22: TOYOPEARL GigaCap Q-650M chromatography operating conditions.

[0497]

[0498]

[0499] Table 23: TOYOPEARL GigaCap Q-650M Eluate Product Quality - Validation Run Summary

[0500]

[0501] The results showed that under optimized conditions, multiple cycles on the GigaCap Q 650M resulted in consistent purity and recovery as confirmed by eluent concentration, SEC-HPLC, RP-HPLC under reducing and non-reducing conditions (Table 19).

[0502] Capto Phenyl HS Hydrophobic Interaction Chromatography (HIC) as a second step

[0503] Below are a number of exemplary methods showing that hydrophobic interaction chromatography (e.g., Capto Phenyl HS) can be used as a second chromatography step (polishing step) for purifying IL-12 fusion polypeptides. Hydrophobic interaction chromatography as a second chromatography step for IL-12 fusion polypeptides has demonstrated high purity and host cell protein (HCP) clearance, and further demonstrated excellent performance in terms of product recovery (approximately 55%) and purity (98.74% monomer, 89.9% NR RP-HPLC, 67% Red RP-HPLC, 92 ppm HCP).

[0504] An exemplary method for hydrophobic interaction chromatography using a linear gradient elution method is described in the table below.

[0505] Table 24: HIC chromatography gradient evaluation operating conditions.

[0506]

[0507] Exemplary methods for hydrophobic interaction chromatography using either stepwise or isocratic conditions are described in the table below.

[0508] Table 25: HIC chromatography isocratic elution operating conditions.

[0509]

[0510]

[0511] Table 26: Summary of HIC isocratic evaluation conditions

[0512]

[0513] Table 27: HIC isocratic elution - data analysis summary

[0514]

[0515]

[0516] The conditions showed high purity and host cell protein (HCP) clearance over multiple column runs (cycling the column 10 times). The conditions resulted in excellent performance in terms of product recovery (approximately 55%) and purity (98.74% monomer, 89.9% NR RP-HPLC, 67% red RP-HPLC, 92 ppm HCP).

[0517] An exemplary method for hydrophobic interaction chromatography is described in the table below.

[0518] Table 28: HIC chromatography operating conditions.

[0519]

[0520]

[0521] TOYOPEARL GigaCap Q-650S anion exchange chromatography as the third step

[0522] Below are several exemplary methods showing 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 concentration in the post-load wash and elution buffers is optimized to select for the optimally phosphorylated IL-12 fusion polypeptide and purify the product from aggregated species and other impurities (host cell proteins and host cell DNA). After loading the column, unbound or flow-through material is washed with equilibration buffer (post-load wash (PLW1)), while less phosphorylated species are removed using PLW2. After elution, the column is stripped with high salt (post-elution wash or PEW) to remove other impurities such as product aggregates, host cell proteins, and host cell DNA.

[0523] For preliminary gradient evaluation, one (1) cycle was performed on TOYOPEARL GigaCap Q-650S resin, and the material was diafiltered into 20mM sodium phosphate pH 7.0 produced by other UF / DF. A 25CV gradient of sodium chloride was used for the elution step, and the loading capacity of the resin was 10.0g / L. The elution peak was classified and the fractions were analyzed by SEC-HPLC, RP HPLC and malachite green assay. The different fractions were merged and submitted to the simulation pool for additional impurity analysis (DNA and HCP). The results of the analysis are summarized in Table 29.

[0524] Table 29: TOYOPEARL GigaCap Q-650S gradient elution - cycle 1 individual fraction analysis summary.

[0525]

[0526] Table 30: TOYOPEARL GigaCap Q-650S Gradient Elution - Cycle 1 Simulated Cell Analysis Summary.

[0527]

[0528]

[0529] Table 31: TOYOPEARL GigaCap Q-650S gradient elution - cycle 2 individual fraction analysis summary.

[0530]

[0531] Table 32: TOYOPEARL GigaCap Q-650S Gradient Elution - Cycle 2 Simulated Cell Analysis Summary.

[0532]

[0533]

[0534] Chromatography was performed on a GigaCap Q-650S using step or isocratic elution. PLW2 = Post-Load Wash 2 (before elution). The purpose of this wash is to remove certain impurities before eluting the product.

[0535] Table 33: Summary of TOYOPEARL GigaCap Q-650S isocratic elution using 27.8% and 33.2% B (Buffer B - 20 mM Tris, 1.0 NaCl pH 7.3) for PLW2 and elution respectively (Cycle 3 - individual fractions analyzed).

[0536]

[0537] Table 34: Summary of isocratic elution with TOYOPEARL GigaCap Q-650S using 27.8% and 33.2% buffer B (containing 1 M NaCl) for PLW2 and elution, respectively (cycle 3 - mock pool analysis).

[0538]

[0539]

[0540] Table 35: Comparison of TOYOPEARL GigaCap Q-650S post-loading wash (PLW2) (before elution) and post-elution wash (PEW) at different sodium chloride molarities.

[0541]

[0542] Table 36: Comparison of elution of TOYOPEARL GigaCap Q-650S at different sodium chloride molarities.

[0543]

[0544]

[0545] 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. Loading capacities up to 20 g / L can be used. Elution collection criteria are set to an absorbance of A280 20 mAU to 500 mAU (A280 0.1 OD to 2.5 OD). PLW2 and elution buffers are formulated, and buffer specifications are more stringent, as variations can impact final polishing steps.

[0546] Figure 17 Shown are multiple cycles of a GigaCap 650S column performed under optimized conditions.

[0547] Table 37: TOYOPEARL GigaCap Q-650S multi-cycle summary.

[0548]

[0549] Table 38: Summary of impurity analysis.

[0550]

[0551] Table 39: GP HPLC analysis

[0552]

[0553] In-process samples were analyzed by malachite green kit assay. The results are shown below.

[0554] Table 40: Summary of phosphorylation analysis

[0555]

[0556] In-process samples from each chromatography step were analyzed at Eurofins using a TDAO (exemplary viral inactivation buffer) detergent detection assay. The results are shown below.

[0557] Table 41: TDAO quantitative summary

[0558]

[0559]

[0560] Summarize

[0561] In some of the manufacturing methods, the purification process 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 before 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.

[0562] The results show that in some of the methods for producing IL-12 fusion polypeptides, the loading capacity of the TOYOPEARL GigaCap Q-650M anion exchange chromatograph at a flow rate of 300 cm / h was approximately 21.4 g / L of resin. The results also show that eluate collection was defined as starting when the upslope A280 nm was ≥ 570 mAU and ending when the downslope A280 nm was ≤ 300 mAU. The yield of the described step exceeded 100%, which may be a result of the titration method used, as octet was used for HCCF titration and nanodrop measurements were used to quantify the product in the eluate.

[0563] For the Capto Phenyl HS chromatography step, a range of binding and elution conditions were evaluated. In some embodiments, a loading capacity of 15.0 g / L of resin can be used. HCP and HMWS clearance data, as well as yield data, indicate that an elution buffer of 20 mM tris, 740 mM ammonium sulfate, pH 7.4, was selected for purification of the IL-12 fusion polypeptide through the final purification process. Optionally, a cleaning procedure including sodium hydroxide, WFI, and guanidine hydrochloride can be implemented, as this can improve resin regeneration.

[0564] Optionally, an intermediate ultrafiltration / diafiltration can be introduced after Capto Phenyl HS chromatography to condition the material before the final polishing step.

[0565] The IL-12 fusion polypeptide was slightly concentrated and the buffer was exchanged to 20 mM tris pH 7.3. Although the retentate showed greater than 98% monomer, high levels of HMWS and LMWS were observed in the buffer wash. In fact, the buffer wash recovery was very low (less than 1% yield), so it is recommended not to include the buffer wash from the intermediate UF / DF in future purification runs until the fractions show the absence of significant HMWS and LMWS.

[0566] The desired product quality was developed using isocratic washing and elution conditions on a TOYOPEARL GigaCap Q-650S chromatograph. A range of sodium chloride molarities in both PLW2 and the elution buffer, as well as collection criteria, were evaluated. The final conditions for the step were determined to be 20 g / L loading, using 274 mM sodium chloride in the PLW2 buffer and 355 mM sodium chloride in the elution buffer. To achieve the desired phosphorylation of the product, eluate collection was defined as starting at an upslope A280 nm ≥ 20 mAU and ending at a downslope A280 nm ≤ 500 mAU. It should be noted that the conductivity specifications for both PLW2 and the elution buffer were very stringent and crucial for running the final polishing step.

[0567] In some embodiments, the manufacturing process includes a filtration step. Planova BioEX filter size results indicate that a 1.0 m2 membrane can be used in a manufacturing facility to process the material in 5 hours at a 1000 L GMP scale.

[0568] 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) into an appropriate buffer (e.g., 20 mM tris, 355 mM sodium chloride pH 7.3) and designated as bulk purified product (BBP).

[0569] The overall cumulative yield of the developed downstream process was 35.1%.

[0570] GP HPLC analysis demonstrated that the IL-12 fusion polypeptide product in the bulk purified product was purified to greater than 98% during this purification process. Non-reducing and reducing RP HPLC also demonstrated high purity, greater than 82% and 55%, respectively. The process also successfully reduced the levels of HCP, DNA, LMWS, and HMWS to acceptable low levels. It was observed that detergents were removed to acceptable levels by the purification process. The bulk purified product exhibited the desired phosphorylation level, demonstrating approximately 7 phosphorus molecules per protein molecule by malachite green assay. Compared to a reference standard batch (C-150921-0079) that exhibited 3X, 4X, 5X, 6X, 7X, 8X, 9X, 10X, 11X, and 13X phosphorylated species, intact ESI-MS observed in the final bulk product. Additionally, fragmentation of 5X, 7X, 9X, 11X, and 13X phosphorylated species was observed in the final bulk product.

[0571] Example 10: Exemplary 500 L scale and 1000 L scale production

[0572] This example demonstrates the production of two batches of IL-12 fusion polypeptide drug substances (a 500 L reference batch and a 1000 L current Good Manufacturing Practice (GMO) batch). Both batches produced comparable high-quality IL-12 fusion polypeptide drug substances. The IL-12 fusion polypeptide drug substances exhibited high purity (e.g., low fragmentation and / or low aggregation), high heterogeneity, appropriate phosphorylation, and / or high activity.

[0573] manufacture

[0574] The IL-12 fusion polypeptide drug substance was manufactured at the following scale:

[0575] 500L scale bioreactor batch (analytical reference standard batch) (P4130826ARS); and

[0576] 1000 L scale bioreactor batch (Good Manufacturing Practice (GMP) batch) (1205114).

[0577] The 500-L 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 prior to distribution and storage. The 1000-L 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.

[0578] In brief, both the 500 and 1000 L scales involve the following steps:

[0579] a) Inoculum expansion, where sufficient viable cells are obtained through serial subculture to serve as inoculum for a production bioreactor. The contents of each vial in the cell bank are thawed and the cells are pelleted by centrifugation. After resuspending the cells in warm growth medium, the cells are transferred to shake flasks. Under an atmosphere containing 5% CO2 in air, the culture is expanded by subculturing in shake flasks and then in roller bottles using growth medium until sufficient cells are produced to inoculate a disposable inoculum bioreactor. The inoculum is further expanded in subsequent inoculum bioreactors until sufficient cells are produced to inoculate a disposable production bioreactor;

[0580] b) Cell culture in a production bioreactor (500 L or 1000 L), where the production bioreactor step is to propagate cell growth under controlled conditions to express the desired quality of recombinant protein, which 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 allowed to reach equilibrium in terms of pH, temperature, and dissolved oxygen tension, and then the cells are aseptically transferred from the inoculation bioreactor to the production bioreactor. Throughout the cell culture process, various additives and feeds are aseptically added to the bioreactor according to feed addition guidelines;

[0581] c) Harvesting removes cells and cell debris and provides a suitable product stream for purification. The production bioreactor is cooled before harvesting, and the culture is then filtered through a depth filter with a nominal pore size range of 9.0 to <0.1 μm to remove cells and cell debris. The clarified supernatant is then sterile filtered through a 0.22 μm filter into a sterile bioprocessing container.

[0582] d) TDAO treatment and anion exchange chromatography (Toyopearl GigaCap Q 650M) wherein N,N-dimethyltetradecamine 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 primary capture step in the downstream purification process. The principle of this step is anion exchange bind and elute chromatography, in which the resin selectively binds the protein, allowing impurities to flow through the column. The processed harvest supernatant is loaded onto an anion exchange column. After loading, the column is washed to allow impurities to be removed, and the bound protein is then eluted as a single fraction using a high salt buffer;

[0583] e) Hydrophobic Interaction Chromatography (HIC, Capto Phenyl, High Sub) where the resin binds the protein, allowing 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 the impurities to be removed, and the bound protein is then eluted as a single fraction by reducing the conductivity of the buffer;

[0584] f) Concentration and diafiltration: The purpose of these steps 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 disposable 30 kDa molecular weight cutoff cassette. The concentrate is then diafiltered in preparation for the next processing step.

[0585] g) Anion exchange chromatography (Toyopearl GigaCap Q 650S) where the resin selectively binds the protein, 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 to allow the impurities to be removed, and the bound protein is then eluted as a single fraction by increasing the buffer conductivity;

[0586] h) Virus reduction filtration, where the product in process is filtered through a small disposable virus retention filter (PlanovaBioEx) to physically remove adventitious viruses (if present). The filter is integrity tested before and after use;

[0587] i) Diafiltration into bulk formulation buffer. The purpose of this step is to exchange the buffer of the in-process product in preparation for the next processing step. The in-process product is processed using an ultrafiltration unit containing a disposable 30 kDa molecular weight cut-off cartridge. The product is then diafiltered into bulk formulation buffer.

[0588] j) Excipient Addition wherein an excipient buffer is added to the in-process diafiltrate in specific ratios to give a final formulation of pH 7.3: 20 mM Tris, 50 mM sodium chloride, 10 mM L-methionine, 150 mM sucrose, 0.02% (w / v) polysorbate 20; and

[0589] k) Bulk Filtration, Filling and Storage The bulk drug substance was filtered through 0.22 μm and aseptically filled into sterile containers (Pall Allegro) and stored. After use, the filter was integrity tested. The drug substance was stored at -65°C or below.

[0590] A summary of batch analysis of the IL-12 fusion polypeptide drug substance is shown in Table 42.

[0591] Table 42: Batch Analysis of IL-12 Fusion Polypeptides

[0592]

[0593]

[0594] CE SDS = capillary electrophoresis sodium dodecyl sulfate; CPA = corrected percent peak area; DNA = deoxyribonucleic acid; ELISA = enzyme-linked immunosorbent assay; HCP = host cell protein; HMW = high molecular weight; LAL = Limulus Amebocyte lysate; Ph. Eur. = European Pharmacopoeia; RPHPLC = reversed-phase high-performance liquid chromatography; SE HPLC = size-exclusion high-performance liquid chromatography; qPCR = quantitative polymerase chain reaction; TAMC = total aerobic count; TYMC = total yeast and mold count; USP = United States Pharmacopoeia.

[0595] 1 The specifications presented are for the release of the GMP 1000 L batch. The pilot 500 L batch was tested to the specifications current at the time of testing.

[0596] 2 Results are obtained from a residual host DNA method evaluation study (consisting of 3 assays). 3 Results are reported from a residual HCP evaluation study (consisting of 3 assays).

[0597] feature

[0598] Results for the IL-12 fusion polypeptide analytical reference standard (500 L batch) and the IL-12 fusion polypeptide GMP (1000 L batch) batches of liquid color, clarity and opalescence, protein concentration, osmolality, and pH all met all specifications. The results for the IL-12 fusion polypeptide analytical reference standard were comparable to those for the GMP drug substance batches. See Table 42.

[0599] Identity and purity

[0600] Reduction RP HPLC was performed on the IL-12 fusion polypeptide reference standard and the IL-12 fusion polypeptide GMP drug substance batch to assess the levels of the pre-peak, main peak, and post-peak as part of batch release testing. The percent main peak, Group A peak area, and Group B peak area for the IL-12 fusion polypeptide GMP drug substance batch and the IL-12 fusion polypeptide reference standard are shown in Table 43. The chromatograms for the GMP batch and the reference standard are presented in Figure 18 middle.

[0601] Table 43: Reduced RP HPLC results for IL-12 fusion polypeptide (ANK-101) reference standard (P4130826ARS) and GMP drug substance batch (1205114).

[0602]

[0603] The major peak is Peak 2 and is labeled as the main peak. Peak Group A and Peak Group B were detected in both samples. The RP HPLC profiles of the GMP drug substance batches were comparable to the reference standard. All results met the current GMP specifications at the time of testing, with a main peak area of ≥60.0% and the % area of the Group A and Group B peaks reported. The identity of the GMP drug substance batches was also confirmed and comparable to the reference standard (Table 42).

[0604] active

[0605] The biological activity (interleukin bioassay) of the GMP batch of IL-12 fusion polypeptide drug substance was 108% relative to the IL-12 fusion polypeptide reference standard. The results for the IL-12 fusion polypeptide reference standard (100% relative to the product standard) and the GMP drug substance batch (108% relative to the IL-12 fusion polypeptide reference standard) were comparable (Table 42).

[0606] purity

[0607] Size heterogeneity (aggregation)-SE HPLC

[0608] SE HPLC was performed on IL-12 fusion polypeptide reference standards and GMP drug substance batches to assess the levels of the 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 presented in Table 44, and the chromatograms are presented in Figure 20 middle.

[0609] Table 44: SE HPLC test results of IL-12 fusion polypeptide (ANK-101) reference standard (P4130826ARS) and GMP drug substance batch (1205114).

[0610]

[0611] Method LOQ = 0.1%, 1 Average of two injections HMW = high molecular weight species; LMW = low molecular weight species; LOQ = limit of quantification

[0612] For both the IL-12 fusion polypeptide reference standard and the IL-12 fusion polypeptide GMP drug substance batch, one high molecular weight species (HMW 1) was detected. When compared to the reference standard, slightly higher levels of the HMW peak were found in the GMP drug substance batch. This is consistent with the results of sedimentation velocity analytical ultracentrifugation (SV-AUC), where higher levels of the HMW species were more consistently detected for the GMP drug substance batch. No LMW species were detected in either sample.

[0613] SE HPLC analysis results showed low aggregation and the GMP drug substance batch was comparable to the reference standard (Table 42).

[0614] Aggregation was measured by anion exchange (AEX) HPLC

[0615] 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 peaks, and basic peaks as part of batch release characterization. The results are shown in Table 45 and the chromatograms are presented in Figure 21 middle.

[0616] Table 45: AEX HPLC test results of IL-12 fusion polypeptide (ANK-101) reference standard (P4130826ARS) and GMP drug substance batch (1205114).

[0617]

[0618] 1 The average of two injections

[0619] Three peaks were detected in both samples. The total relative percentages of Main Peak 1, Main Peak 2, and the acidic peak were calculated. When compared to the reference standard, Main Peak 1 was slightly higher and Main Peak 2 was slightly lower for the GMP drug substance batch. However, the sum of the main peaks was comparable between samples. This may be related to the slightly lower phosphorylation level of the GMP drug substance batch determined by malachite green phosphoprotein estimation. However, the sum of the main peaks was comparable between samples. The AEX HPLC analysis results showed low aggregation, and the GMP drug substance batch was comparable to the reference standard by AEX HPLC.

[0620] Phosphoprotein Phosphate Estimation Assay (Malachite Green)

[0621] The phosphorylation levels of 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 the findings from reverse phase high performance liquid chromatography (RP HPLC) by electrospray ionization mass spectrometry (ESI-MS) to reduce tryptic peptide patterns and mass isomer heterogeneity. The manufactured IL-12 fusion polypeptide showed an appropriate degree of phosphorylation (Table 42).

[0622] Fragmentation was measured by reducing capillary electrophoresis sodium dodecyl sulfate assay (CE-SDS)

[0623] The electrophoretic patterns of the IL-12 fusion protein reference standard and the GMP drug substance batch were evaluated using capillary electrophoresis sodium dodecyl sulfate (CE-SDS) under reducing conditions as part of batch release testing. The percent purity of the GMP drug substance batch and the reference standard were 97.4% and 95.4%, respectively (Table 42). The chromatograms of the GMP batch and the reference standard are presented in Figure 19 middle.

[0624] The main peak for both samples met the current GMP specification of ≥90.0% at the time of testing. An additional minor peak (Peak 4) was detected in the reference standard, and an additional minor peak (Peak 6) was detected in the GMP drug substance batch. However, the profiles were comparable with the variation of the minor peak below the limit of quantification (LOQ). All results met the current GMP specifications 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).

[0625] Impurities (Process)

[0626] As part of batch release testing, the IL-12 fusion polypeptide reference standard and GMP drug substance batches were tested by quantitative polymerase chain reaction (qPCR) on Chinese hamster ovary (CHO) deoxyribonucleic acid (DNA) and by CHO host cell protein (HCP) enzyme-linked immunosorbent assay (ELISA). The levels of process-related impurities in the GMP drug substance batches were comparable to those in the reference standard (Table 42).

[0627] Security

[0628] Bioburden and endotoxin testing were performed on the IL-12 fusion polypeptide reference standard and GMP drug substance batches as part of batch testing. Safety results for the GMP drug substance batches were comparable to the reference standard (Table 42).

[0629] Example 11: Characterization of Exemplary IL-12 Fusion Polypeptide Drug Substances

[0630] This example provides exemplary IL-12 fusion polypeptide drug substance characterization. This example demonstrates that IL-12 fusion polypeptides have a phosphorylation degree of 7.1 or 8.

[0631] ESI-MS mass spectrometry analysis

[0632] Relative molecular mass was determined by ESI-MS. Due to the presence of a variety of different sialylated glycans (N-linked and O-linked glycans) and phosphorylation sites, IL-12 fusion polypeptide drug substances contain a high degree of heterogeneity in product variants. Therefore, the mass of the intact IL-12 fusion polypeptide drug substance cannot be directly determined by ESI-MS. Instead, heterogeneity must be reduced by, at a minimum, removing glycans (deglycosylation) using an enzyme cocktail containing an N-glycosidase (PNGaseF), a sialidase (SialExo), and an O-glycanase (OglyZOR). This was performed under reducing and denaturing conditions to increase the exposure of these post-translational modifications to the enzymes. Samples of the deglycosylated product were prepared with and without dephosphorylation by lambda phosphatase and analyzed by online desalting ESI-MS. Using this method, spectra were obtained that confirmed the primary mass of the deglycosylated product with and without phosphorylation. To obtain additional information on the distribution of differentially phosphorylated species / low intensity species, a sample of the deglycosylated product sample was also analyzed by a second ESI-MS method and on-line RP HPLC to provide the necessary resolution.

[0633] ESI-MS results of online desalting

[0634] A deglycosylated IL-12 fusion polypeptide drug substance in-process (pre-excipient) sample was analyzed by ESI-MS using online desalting. The deconvoluted spectra are presented in Figure 22 The major masses detected in the in-process samples of the reference and GMP batches of the deglycosylated IL-12 fusion polypeptide drug substance were 64,151 Da and 64,152 Da (species 3), respectively, which are consistent with the expected theoretical mass (64,154 Da) of the deglycosylated IL-12 fusion polypeptide drug substance with nine phosphorylations. The theoretical mass is based on the theoretical amino acid sequence of the IL-12 fusion polypeptide drug substance, which has fully reduced disulfide bonds and all asparagine residues at potential N-linked glycosylation sites converted to aspartic acid due to enzymatic deglycosylation. A total of five species were assigned masses, with masses consistent with varying degrees of phosphorylation (theoretical mass difference (delta mass) for phosphorylation is +80 Da).

[0635] ESI-MS spectra were visually comparable, confirming similar levels of phosphorylated species for the GMP drug substance batch and the reference standard batch (a representative in-process, pre-excipient spiked sample). This was consistent with findings from malachite green phosphoprotein phosphate estimation and RP HPLC tryptic peptide mapping.

[0636] A deglycosylated and dephosphorylated IL-12 fusion peptide drug substance in-process sample was analyzed by online desalting ESI-MS. Dephosphorylation was incomplete but sufficient to reveal species consistent with the underlying protein mass. The deconvoluted spectrum is presented in Figure 22 The main masses detected in the deglycosylated and dephosphorylated IL-12 fusion polypeptide drug substance in-process samples of the reference and GMO batches were 63,513 Da and 63,514 Da (species 2), respectively, which is consistent with the expected theoretical mass (63,514 Da) of the deglycosylated IL-12 fusion polypeptide drug substance with one phosphorylation. For the two in-process samples of the reference and GMO batches, 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 (theoretical mass 63,434 Da). Species 3 was observed in both samples, which is consistent with the deglycosylated IL-12 fusion polypeptide drug substance with two phosphorylations or one hexose, which is indicative of glycation (glycation was confirmed by RPHPLC reduced tryptic peptide mapping). The ESI-MS spectra of the reference and GMO batch in-process samples were visually comparable.

[0637] ESI-MS results of online RP HPLC

[0638] Deglycosylated IL-12 fusion peptide reference standard and GMP drug product batches were analyzed by ESI-MS with online RP HPLC. Total ion current chromatograms are presented in Figure 23 , and the deconvoluted spectrum is presented in Figure 24 The major masses detected for the reference standard and GMP drug product batches were 64,437.6 Da and 64,437.1 Da, respectively, which are consistent with the mass of the IL-12 fusion polypeptide drug substance (theoretical mass 64,437.1 Da) without N-linked glycans but with O-linked glycans (HexHexNAc) and eight phosphorylations. This suggests that either enzymatic processing of the O-glycans in this assay is incomplete or that background matrix effects are reduced by online RP-HPLC, resulting in better detection of O-glycosylated species using this method. The other masses are consistent with varying degrees of phosphorylation, with or without individual O-linked glycans. The occupancy and levels of phosphorylation observed between the two batches were comparable and consistent with those determined by RP-HPLC reduced tryptic peptide mapping and malachite green phosphoprotein phosphate estimation.

[0639] ESI-MS of online RP HPLC revealed that the phosphorylation spectrum of the product (5 to 12 phosphorylations; the main species has 8 phosphorylations) is larger than the phosphorylation spectrum determined by online desalting ESI-MS (7 to 11 phosphorylations; the main species has 9 phosphorylations). This may be due to differences in species resolution caused by the different mass spectrometers used for each method and differences in sample matrix interference (better removal of interfering / ion suppression entities by RP HPLC compared to desalting alone). Overall, both methods confirmed that the quality of the batches was as expected, and the spectra of the product variants between the two batches were comparable.

[0640] RP HPLC-MS and MS / MS reduced tryptic peptide mapping

[0641] The test sample was denatured in guanidine hydrochloride and reduced with tris(2-carboxyethyl)phosphine (TCEP). The sample buffer was subsequently exchanged into urea / TCEP buffer for trypsin digestion to maintain both enzyme activity and the solubility of the molecule. The trypsin digestion sample was then used and RP HPLC was used for separation. A combination of mass spectrometry and ultraviolet (UV) detection (λ 210 nm and λ 280 nm) was used.

[0642] 210 nm, 280 nm and total ion count (TIC) spectra were generated. TIC chromatograms of the IL-12 fusion polypeptide reference standard and the GMP drug substance batch were visually comparable.

[0643] Sequence coverage

[0644] Signals from the acquired mass spectra were matched to theoretical tryptic digest patterns of the IL-12 fusion polypeptide drug substance. Sequence coverage of 100% for the IL-12 fusion polypeptide reference standard and the GMP drug substance batch was supported by MS confirmation and MS / MS assignment of intact, partially digested (mis-cleaved) peptides using a combination of untreated and dephosphorylated samples.

[0645] N-terminal sequence

[0646] N-terminal sequence verification was performed on the IL-12 fusion peptide 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.

[0647] C-terminal sequence

[0648] C-terminal sequence verification of the IL-12 fusion peptide reference standard and GMP drug substance batches was performed using mass spectrometry and tandem mass spectrometry detection. Under dephosphorylation conditions, the C-terminal peptide (VMSYLNASGGGGEGGGGSEEGGGGSEEGGGGSEEGGGGSEEGGGGSEEGGGGSEEGGGGSEEGGGGSEEGGGGGS, P59) was detected as both unmodified and monophosphorylated peptides in both samples.

[0649] Post-translational modification

[0650] For IL-12 fusion peptide reference standards and GMP drug substance batches, the approximate amount of potential post-translational modifications is assigned by comparing the ion intensity from the modified peptide with the ion intensity from the unmodified peptide. The results must be regarded as approximate, as the chromatogram and intensity of low-abundance modified peptides cannot be assumed to have the same ionization characteristics as the unmodified peptide.

[0651] Deamidation

[0652] Ions consistent with potential deamidated variants of the IL-12 fusion polypeptide reference standard and GMP drug substance batches were observed. The putative deamidated peptides had a molecular weight 1 Da higher than the corresponding intact peptide due to replacement of the amide on the asparagine (N) or glutamine (Q) residues with a carboxyl group (conversion to aspartic acid / isoaspartic acid and glutamic acid residues, respectively). In general, peptides prone to deamidation have a high incidence of N and Q residues flanked by serine (S) and glycine (G) residues, suggesting that they stabilize intermediates in the deamidation reaction.

[0653] Ions consistent with potential deamidation of P 17 were detected at 1% in both samples. Deamidation levels in GMP drug substance batches were comparable to the reference standard.

[0654] Oxidation

[0655] Ions consistent with potential oxidized variants of the IL-12 fusion polypeptide reference standard and GMP drug substance batches were observed. Assuming that either methionine (M) or tryptophan (W) was oxidized, the putative oxidized peptide had a molecular weight 16 Da higher than the corresponding intact peptide.

[0656] Three signals consistent with potential oxidation were detected in both samples. The oxidation levels detected in the GMP drug substance batches were comparable to the reference standard.

[0657] Glycosylation

[0658] The occupancy and heterogeneity of potential glycosylation sites were assessed using tryptic digests of an IL-12 fusion polypeptide reference standard and a GMP drug substance batch. The theoretical amino acid sequence of the IL-12 fusion polypeptide drug substance displayed seven sequence motifs commonly associated with N-linked glycosylation (Asn-X-Ser / Thr, where X is any residue other than 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 at Asn103, Asn200, Asn281, Asn392, and Asn406. No glycosylation was detected at the potential N-linked glycosylation sites Asn113 and Asn516. There was also evidence of attachment of O-linked glycans. Although it is known that O-linked glycans are typically attached via the side groups (hydroxyl groups) of serine and threonine residues, the exact site of O-linked glycosylation cannot be predicted from the sequence. One peptide containing O-linked glycosylation was identified from tryptic digestion data. The N-linked and O-linked glycosylation site occupancy of the GMP drug substance batch was comparable to that of the reference standard.

[0659] Phosphorylation

[0660] The IL-12 fusion polypeptide drug substance is believed to be phosphorylated by the co-expressed human kinase Fam20C. Phosphorylation of the IL-12 fusion polypeptide drug substance by the human kinase Fam20C was assessed by reduced tryptic peptide mapping RP HPLC-MS. The estimated levels of phosphorylation at each site are shown in Table 46.

[0661] Table 46: Estimated Phosphorylation Occupancy (%) for an Exemplary IL-12 Fusion Polypeptide Reference Standard (P4130826ARS) and a GMP Drug Substance Batch (1205114)

[0662]

[0663]

[0664] For both the GMP drug substance batch and the reference standard, phosphorylation sites detected were Ser43, Ser154, Ser168, Ser281, Ser306, Ser311, Ser316, Ser406, and Ser481. 1% phosphorylation was detected in the reference standard at Ser365, whereas it was not detected in the GMP drug substance batch. For both batches, phosphorylation was not detected at the potential phosphorylation sites Ser233, Ser491, Ser498, Ser505, Ser512, Ser520, Ser527, Ser534, and Ser541.

[0665] Overall, phosphorylation levels of GMP drug substance batches were comparable to the reference standard, consistent with findings from malachite green phosphoprotein phosphoester estimation and ESI-MS mass isoform heterogeneity.

[0666] Glycosylation

[0667] Additional glycation occurs most readily at the N-terminal α-amino group of proteins or at the ε-amino groups of lysine residues throughout the protein sequence.

[0668] The reduced tryptic peptide map MS and MS / MS data were searched to determine if there was evidence of hexose attachment, indicating glycation. Given that trypsin does not cleave at glycated lysine residues, a data search was performed on tryptic peptides with internal lysine residues (i.e., peptides that were not completely cleaved) and a single hexose with an additional mass (162 Da). Due to reliance on partially digested peptides, it was not possible to determine glycation levels solely through peptide maps. Therefore, only these data indicated that glycation was detected, and the precise mass and level were not reported in this study.

[0669] Glycation testing of GMP drug substance batches is comparable to reference standards.

[0670] neutral oligosaccharides

[0671] HPLC and LC-MS methods are used to determine neutral and charged N-linked oligosaccharide profiles. N-linked oligosaccharides are released by incubation with the enzyme peptide-N-glycosidase F (PNGase F) under denaturing and reducing conditions. Protein is removed and the released oligosaccharides are prepared for neutral and charged analysis. The released glycans are labeled with the fluorophore 2-aminobenzamide (2-AB) and incubated with α-sialidase to remove terminal sialic acid. Neutral profile - N-linked oligosaccharides are released under native conditions by incubation with recombinant peptide-N-glycosidase F (PNGaseF). Protein is removed by precipitation and the released oligosaccharides are hydrolyzed. Oligosaccharides are labeled with the fluorophore 2-aminobenzamide (2-AB) and treated with α-sialidase before analysis. The glycan profile and peaks are quantified by fluorescence peak area and identified by LCMS.

[0672] The N-linked oligosaccharide profiles of IL-12 fusion peptide reference standards and GMP drug substance batches were determined using LC-MS of fluorescently labeled glycans released by PNGase F. The chromatograms are presented in Figure 25 middle.

[0673] The proposed oligosaccharide structures were assigned based on accurate mass measurements. Further confirmation was performed by retention time comparison with N-glycan standards and, where appropriate, IgG IAC was used to aid identification.

[0674] The detected N-glycans were consistent with a biantennary complex glycan structure that was primarily core-fucosylated, with varying levels of terminal galactosylation, as expected for the GS-CHO cell line. The major 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 of the N-linked oligosaccharide analysis were consistent with those from the peptide map.

[0675] Some variation in the levels of minor glycans was observed, but overall, the N-linked oligosaccharide profiles of IL-12 fusion polypeptide GMP drug substance batches were considered comparable to the reference standard due to the absence of additional glycan structures compared to the current reference standard and similar levels of major glycans.

[0676] Charged oligosaccharides

[0677] Charged Profile - 2-AB labeled glycans were separated based on charge by HPLC. Sialyl glycans were identified by comparison with the retention times of mono-sialylated, di-sialylated, tri-sialylated, 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.

[0678] Charged N-linked oligosaccharide profiles of an IL-12 fusion polypeptide reference standard and a GMP drug substance batch were analyzed by hydrophilic interaction liquid chromatography (HILIC) with fluorescently labeled N-linked glycans. The identities of the reported charged groups were confirmed by comparison with a test sample treated with α-sialidase. Neutral mono-, di-, and tri-sialylated glycans were detected in both the IL-12 fusion polypeptide GMP drug substance batch and the reference standard, and the charged oligosaccharide profiles were comparable.

[0679] Determination of the binding kinetics and strength of aluminum hydrogel / IL-12 fusion peptide drug substances

[0680] The IL-12 fusion peptide drug substance manufacturing process has been developed and scaled up to produce a 500L toxicology batch (P4130826, the current reference standard) and a 1000L GMP batch (1205114). Samples from each batch were characterized using aluminum hydrogel binding kinetics and strength assays. Based on these results, the IL-12 fusion peptide drug substance remained comparable throughout the manufacturing process development.

[0681] The purpose of this assay is to measure the kinetics and binding strength of an IL-12 fusion polypeptide drug substance (IL-12-ABP) on aluminum hydrogels (aluminum hydroxide particles; alum). The IL-12-ABP protein contains a C-terminal fusion with a phosphorylated aluminum binding peptide (ABP) and is expected to form a stable complex with the aluminum hydrogel via a ligand exchange reaction between the phosphoserine in the peptide and surface hydroxyl groups on the aluminum hydrogel. To test the stability (strength) of this complex, the bound protein is exposed to phosphate and human serum, simulating in vivo conditions, and the weakly bound protein is eluted from the aluminum hydroxide. In the absence of aluminum hydrogel, IL-12-ABP is included in the assay as a non-binding protein stability control.

[0682] IL-12-ABP (IL-12 fusion polypeptide drug substance) was bound to aluminum hydrogel at a final concentration of 0.25 mg / mL protein and 2.5 mg / mL aluminum hydroxide (1:10 ratio) at variable time intervals of 5, 10, and 30 minutes by mixing the two components in phosphate-free IL-12-ABP formulation buffer. To test the aluminum hydrogel binding kinetics, samples at different time intervals were centrifuged to pellet all alum-bound protein particles. The protein content was determined by IL-12p70-specific sandwich ELISA assay (ELISA MAX TM Deluxe Set Human IL-12 (p70); BioLegend) (Table 47) quantified free (unbound) IL-12-ABP in the supernatant.

[0683] Table 47: Comparison of binding kinetics of IL-12-ABP (IL-12 fusion polypeptide drug substance (ANK-101)) to Aluminum Hydrogel - Lot No. P4130826 (current reference standard) and 1205114 (GMP Lot No.).

[0684]

[0685] *All samples were diluted to 10 μg / mL before reading

[0686]

[0687] *All samples were diluted to 10 μg / mL before reading

[0688] IL-12-ABP was captured using a mouse IgG1 (R&D Systems) monoclonal antibody. Next, a biotinylated mouse monoclonal anti-human IL-12 (p70) detection antibody was added, followed by avidin-HRP reagent, followed by TMB substrate, which produces a blue color proportional to the concentration of IL-12 (p70) present. Each plate included an 8-point free IL-12-ABP standard curve (each point was repeated), and alum-bound IL-12-ABP conditions were included and analyzed in triplicate. To test the binding strength of the IL-12-ABP interaction with alum, alum-bound protein samples were diluted in 1 mM phosphate and 40% human serum at 5, 10, and 30 minute intervals and incubated at 37°C for 24 hours. At 2 and 24 hours, samples were removed and centrifuged to precipitate the aluminum hydrogel particles. Free (unbound) IL-12-ABP in the supernatant was quantified as described above.

[0689] Table 48: Comparison of binding strength of IL-12 ABP (IL-12 fusion polypeptide drug substance (ANK-101)) to aluminum hydrogel - batches P4130826 (current reference standard) and 1205114 (GMP batch).

[0690]

[0691] * IL-12-ABP and aluminum water After incubation for 5, 10, or 30 minutes (association time), the three formulations were then incubated in 1 mM phosphate, 40% human serum for 24 hours; all samples were diluted to 10 μg / mL before reading.

[0692]

[0693] * IL-12-ABP and aluminum water After incubation for 5, 10, or 30 minutes (association time), the three formulations were then incubated in 1 mM phosphate, 40% human serum for 24 hours; all samples were diluted to 10 μg / mL before reading.

[0694] Example 12: Impurities - Permitted Daily Intravenous Exposure (PDE IV )

[0695] The examples demonstrate 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. It is expected that high molecular weight compounds (PDMS, pluronic, TDAO) will be removed from the process during the three bind and elute chromatography unit operations. The manufacturing process also removes Fam20.

[0696] In this example, process-related impurities, product-related impurities, and contaminants that may be present in the IL-12 fusion polypeptide drug substance were measured.

[0697] The following additives are classified as process-related impurities that have been identified as a concern for patient safety and are not controlled during the manufacture of the IL-12 fusion polypeptide drug substance or as part of release testing. A toxicology evaluation has been performed to consider the risk to patients from exposure to these components.

[0698] Tropolone: Tropolone is added as a nutritional supplement to the production phase of the cell culture process. Tropolone is a lipophilic iron chelator that improves iron transport into cells, enabling cell growth and maintaining cell viability.

[0699] Pluronic: Pluronic is a component of the base powder used to prepare inoculum and production-phase bioreactor culture media. The purpose of this chemical is to enhance cell membrane stability during cell culture.

[0700] Polydimethylsiloxane (PDMS) and Octamethylcyclotetrasiloxane D4: Polydimethylsiloxane (PDMS) and Octamethylcyclotetrasiloxane D4 are components of simethicone, the active ingredient in Antifoam C Emulsion. During manufacturing, Antifoam C Emulsion is typically added to cell cultures to control or suppress foaming in production bioreactors. Addition is variable and depends on the degree of foaming.

[0701] N,N-Dimethyltetradecamine N-oxide (TDAO): N,N-Dimethyltetradecamine N-oxide (TDAO) is a detergent added to the clarified harvest supernatant for viral inactivation. This supplement is added to the harvested cell culture medium prior to the first chromatography step.

[0702] Fam20C: Human Fam20C is an endogenous Golgi-localized serine / threonine kinase that phosphorylates serine and threonine motifs on proteins. It is co-expressed with ANK-101 and phosphorylates an alum-conjugated peptide drug substance.

[0703] The permitted daily intravenous exposure (PDE) for the identified impurities tropolone, pluronic, PDMS, octamethylcyclotetrasiloxane D4, TDAO, and Fam20C has been determined through toxicological evaluation. IV ) limits are summarized in Table 49.

[0704] Table 49: PDE IV and its potential adverse effects on potential impurities of the IL-12 fusion polypeptide drug substance.

[0705]

[0706] PDE IV = daily permitted intravenous exposure; PDMS = polydimethylsiloxane; TDAO = N,N-dimethyltetradecamine N-oxide

[0707] Risk Assessment of the Clearance Rates of TDAO, Tropolone, Pluronic, PDMS, and Octamethylcyclotetrasiloxane D4

[0708] Based on the defined PDE IV A risk assessment calculation was performed to assess the risk of these potential impurities remaining in the downstream process by determining the level of each impurity that would be present in a dose of the IL-12 fusion peptide drug substance if no claim of elimination of each impurity was made throughout the manufacturing process (maximum transport model). This assessment was based on the manufacturing process of the IL-12 fusion peptide drug substance batches and adopted a conservative approach.

[0709] To ensure that the compound of interest is below the PDE in the IL-12 fusion polypeptide drug substance IV , many log clearances are required. If possible, it is best to demonstrate a 90% safety factor for clearance, i.e., the amount of compound of interest remaining is the PDE IV 10% or less of the total. Ensure potential impurity levels are below the PDE IV The total number of complete log clearances required for both 5% and 90% safety factors is summarized in Table 50.

[0710] Table 50: Summary of Compound Evaluation

[0711]

[0712] PDE IV = daily permitted intravenous exposure; PDMS = polydimethylsiloxane; TDAO = N,N-dimethyltetradecamine N-oxide

[0713] Study on the clearance rate of TDAO

[0714] Based on the defined PDE IV, performed a risk assessment calculation that calculated how much TDAO would be present in a dose of an IL-12 fusion polypeptide drug substance if no claim of TDAO removal was made throughout the manufacturing process. The assessment took a conservative approach based on the batch manufacturing process for the IL-12 fusion polypeptide drug substance and considered the following factors: (1) the amount of TDAO contained in the culture (assuming that the total amount added to the bioreactor did not change from the amount present after harvest), (2) the product titer on the day 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 of IL-12 fusion polypeptide drug product and a 55 kg patient).

[0715] Based on this scenario, the amount of TDAO present in the 0.02 mg / kg dose is summarized in Table 51.

[0716] Table 51: TDAO safety margin calculation

[0717]

[0718] 1 Calculations were made based on the amount of TDAO added to the harvested cell culture medium.

[0719] PDE IV = daily permitted intravenous exposure; TDAO = N,N-dimethyltetradecamine N-oxide

[0720] To ensure that TDAO levels are below the PDE in IL-12 fusion peptide drug substances IV , many log clearances are required. If possible, it is best to demonstrate a 90% safety factor for clearance, i.e., the amount of compound of interest remaining is 10% or less of the PDE IV. Furthermore, the total number of complete log clearances required to ensure both TDAO levels below the PDE IV and a 90% safety factor is summarized in Table 52.

[0721] Table 52: Minimum Log Clearance of TDAO

[0722]

[0723] PDE IV = daily permitted intravenous exposure; TDAO = N,N-dimethyltetradecamine N-oxide; NA = not applicable

[0724] Determination of TDAO in in-process samples

[0725] TDAO was quantified by monitoring two MS / MS mass transitions using reversed-phase liquid chromatography-mass spectrometry (RP-LC-MS / MS) detection. A calibration curve for the determination of detergents by LC-MS / MS 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 concentration of the analyte samples.

[0726] To confirm the clearance of the compound TDAO during the downstream process, intermediate samples from pilot batches and good manufacturing practice (GMP) batches were submitted for LC-MS / MS testing of TDAO. In addition, the buffer matrix of the samples was also analyzed. All samples were processed / analyzed in duplicate to have confirmed results for each sample (one of the two replicates was injected twice to check the repeatability of the injection / analysis run).

[0727] All samples analyzed showed TDAO levels below the limit of detection (LOD) of 0.3 mg / L. Capto Phenyl HS eluate samples were submitted for pilot and GMP batches, and the results showed that TDAO was cleared (below the LoD) after two chromatographic steps and an intermediate ultrafiltration / diafiltration (UF / DF). Because TDAO levels were found to be below the LoD, a high safety margin for TDAO clearance is provided in the current downstream process.

[0728] Conclusions of the impurity transport model for TDAO, tropolone, pluronic, PDMS, and octamethylcyclotetrasiloxane D4

[0729] It is expected that the desired logarithmic clearance numbers will be achieved during the multiple rounds of bind and elute chromatography and two diafiltrations that make up the process. It is expected that lower molecular weight compounds (octamethylcyclotetrasiloxane D4, tropolone) will be removed from the process during the three bind and elute chromatography unit operations. It is expected that nearly all of the remaining compounds in the permeate will be removed during the concentration and diafiltration stages of the intermediate UF / DF step.

[0730] It is expected that higher molecular weight compounds (PDMS, Pluronic, TDAO) will be removed from the process during the three bind and elute chromatography unit operations. Any remaining compounds, as long as they are below their critical micelle concentrations, will behave similarly to the lower molecular weight compounds on the intermediate UF / DF. Specifically, TDAO levels were measured after Capto Phenyl HS chromatography on different in-process samples from three different batches and were found to be below the detection limit.

[0731] Quantification of Fam20

[0732] The amount of Fam20C was monitored by LC-MS / MS using a multiple standard addition method. Fam20C was spiked into the analyte at three levels. The amount of Fam20C in each sample was measured using LC-MS / MS monitoring of tryptic peptide 31. A linear regression was then performed on the measured levels in the spiked samples, and the original unknown amount of Fam20C was estimated by solving the fitted straight line equation for y=0. The Fam20C quantification results for the pilot and GMP batches are summarized in Table 53.

[0733] Table 53: Fam20C quantification

[0734]

[0735] GMP = Good Manufacturing Practice

[0736] When considering the maximum dose of IL-12 fusion polypeptide drug substance (1.25 mg / d), the amount of Fam20C per mg of IL-12 fusion polypeptide drug substance in the GigaCap Q-650M was measured relative to the PDE IV At the end of the purification process, the amount of Fam20C / mg of IL-12 fusion polypeptide drug substance was further reduced by two orders of magnitude, indicating that the purification process was able to remove Fam20C.

[0737] Equivalent

[0738] 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.It is intended that the scope of the invention be limited not by the above description, but rather by that set forth in the following claims.

Claims

1. A method for producing a phosphorylated form of a fusion polypeptide, wherein the fusion polypeptide comprises (a) an immunomodulatory polypeptide comprising an immunoagonist portion; and (b) a metal hydroxide binding polypeptide, Its amino acid sequence contains multiple phosphorylation sites, so that the fusion polypeptide can adopt phosphorylated and non-phosphorylated forms. The method includes the step of co-expressing the fusion polypeptide and a kinase that phosphorylates the fusion polypeptide in a host cell such that the fusion polypeptide and the kinase are expressed at a ratio ranging from about 4:1 to about 10:

1.

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

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

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

5. The method of claim 1, wherein the step of co-expressing comprises expression from a transposon plasmid. The method of claim 5 , wherein the transposon plasmid is a DNA plasmid.

7. The method of claim 5, wherein the cells are further transfected with a nucleotide sequence encoding a transposase.

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

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

10. The method of claim 7, wherein the nucleotide sequence encoding the transposase 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. The method of claim 9, wherein the integration results in a single copy integration in each genomic locus.

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

1.

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

15. The method of claim 14, wherein the Fam20C kinase has an amino acid sequence that is at least 80% identical to the amino acid sequence 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 immune agonist portion comprises a first portion and a second portion or a functional fragment thereof.

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. 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 the amino acid sequence of SEQ ID NO: 2, and the second portion comprising the IL12 portion has an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:

3.

20. A method for producing a highly purified preparation of the phosphorylated form of a fusion polypeptide from a cell extract comprising the phosphorylated form of the fusion polypeptide, The fusion polypeptide comprises (a) an immunomodulatory polypeptide comprising an immunoagonist portion; and (b) a metal hydroxide binding polypeptide, Its amino acid sequence contains multiple phosphorylation sites, allowing the fusion polypeptide to adopt phosphorylated and non-phosphorylated forms; and further wherein the cell extract is from cells expressing the fusion polypeptide and the kinase at 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 of claim 20, wherein the method comprises one or more purification steps selected from the group consisting of: anion chromatography step; and Hydrophobic interaction chromatography step.

22. A method for producing a highly purified preparation of the phosphorylated form of a fusion polypeptide from a cell extract comprising the phosphorylated form of the fusion polypeptide, The fusion polypeptide comprises (a) an immunomodulatory polypeptide comprising an immunoagonist portion; and (b) a metal hydroxide binding polypeptide whose amino acid sequence comprises multiple phosphorylation sites, such that the fusion polypeptide can adopt phosphorylated and non-phosphorylated forms; and further wherein the cell extract is derived from cells expressing the fusion polypeptide and the kinase at a ratio ranging from about 4:1 to about 10:1, such that the phosphorylated form is produced; The method comprises one or more purification steps selected from the group consisting of: anion chromatography step; and Hydrophobic interaction chromatography step.

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. 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 method of claim 21 or 22, wherein the purification step comprises: i) a first anion chromatography step; ii) a hydrophobic interaction chromatography step; as well as iii) Second anion chromatography step.

26. 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 utilizes a higher salt concentration to elute the phosphorylated fusion polypeptide.

27. 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 utilizes a lower ammonium sulfate concentration to elute the phosphorylated fusion polypeptide.

28. 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 utilizes a higher salt concentration to elute the phosphorylated fusion polypeptide.

29. The method of claim 21 or 22, wherein the purification step comprises a preliminary viral inactivation step.

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

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

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

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

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

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

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

37. The cell of claim 36, wherein promoters of different strengths are used to produce 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 the SV40 promoter or the Ubc promoter.

40. A preparation of cultured mammalian cells as claimed in claim 35.

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

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

43. The preparation of claim 42, wherein said expression of said fusion polypeptide and said kinase is reasonably comparable to said expression of a monoclonal preparation of the same cells.

44. A method comprising the steps of: 1) Cultivating engineered mammalian cells expressing: i) a first nucleotide sequence encoding a fusion polypeptide, wherein the fusion polypeptide comprises: a) an immunomodulatory polypeptide comprising an immunoagonist portion; and b) a metal hydroxide binding polypeptide whose amino acid sequence contains multiple phosphorylation sites, so that the fusion polypeptide can adopt phosphorylated and non-phosphorylated forms ii) a second nucleotide sequence encoding a kinase; The first nucleotide sequence and the second nucleotide sequence are in cis with each other and are each controlled by a regulatory nucleotide sequence that controls expression, such that the fusion polypeptide is expressed at a ratio of about 4:1 to about 10:1 of the ratio of expression of the kinase II) Purifying the phosphorylated fusion polypeptide without an affinity chromatography step.

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

46. In a method for purifying a phosphorylated form of a fusion polypeptide from a cell extract comprising the phosphorylated form of the fusion polypeptide, The fusion polypeptide comprises (a) an immunomodulatory polypeptide comprising an immunoagonist portion; and (b) a metal hydroxide-binding polypeptide, the amino acid sequence of which contains multiple phosphorylation sites, so that the fusion polypeptide can adopt phosphorylated and non-phosphorylated forms, the improvement comprising: The purification was performed without an affinity chromatography step.

47. The improvement of claim 46, wherein the improvement further comprises: The purification is performed from a cell extract prepared from a culture of the mammalian cell according to claim 35.

48. In a method for purifying a phosphorylated form of a fusion polypeptide from a cell extract comprising the phosphorylated form of the fusion polypeptide, The fusion polypeptide comprises (a) an immunomodulatory polypeptide comprising an immunoagonist portion; as well as (b) a metal hydroxide binding polypeptide, the amino acid sequence of which contains multiple phosphorylation sites, so that the fusion polypeptide can adopt phosphorylated and non-phosphorylated forms, the improvement comprising: The purification is performed by a method comprising one or more steps selected from the group consisting of: anion chromatography step; and Hydrophobic interaction chromatography step.

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