Granulocyte-macrophage colony-stimulating factor production

Abstract

The present disclosure relates to a manufacturing process for sargramostim, which improves production efficiency and yield.

Description

[Technical Field]

[0001] Priority This application claims priority to and benefits of U.S. Provisional Patent Application No. 63 / 122,593 filed on 8 December 2020 and U.S. Provisional Patent Application No. 63 / 271,444 filed on 25 October 2021, each of which is incorporated herein by reference in its entirety.

[0002] Technical field This invention broadly relates to methods for improving and increasing the production of granulocyte-macrophage colony-stimulating factor (GM-CSF).

[0003] Description of the electronically submitted text file This application includes an ASCII-formatted sequence listing submitted electronically via EFS-Web together with this application. The above ASCII record, created on December 6, 2021, is named PNR-004PC_SequenceListing_ST25.txt and is 4,096 bytes in size. The above sequence listing is incorporated herein by reference in its entirety. [Background technology]

[0004] Colony-stimulating factors (CSFs) refer to a family of four glycoproteins that control and regulate cell production from widely dispersed, isolated myeloid cell deposits. These include granulocyte-macrophage CSF (GM-CSF), granulocyte colony CSF (G-CSF), macrophage colony CSF (M-CSF), and pluripotent colony-stimulating factor (IL-3). These lymphokines can induce progenitor cells present in the bone marrow to differentiate into specific types of mature blood cells. The specific type of mature blood cell that arises from the progenitor cells is determined by the type of CSF present. See Metcalf D. Cancer Immunol Res. 2013, 1(6):351-356.

[0005] GM-CSF is a hematopoietic growth factor that regulates the production, migration, proliferation, differentiation, and function of hematopoietic cells. In response to inflammatory stimuli, GM-CSF is released by various cell types, including T lymphocytes, macrophages, fibroblasts, and endothelial cells. Subsequently, GM-CSF activates and enhances the production and survival of neutrophils, eosinophils, and macrophages. Naturally occurring GM-CSF is usually produced in vitro near the site of action where it regulates the proliferation, differentiation, and survival of hematopoietic progenitor cells, but in circulating blood, GM-CSF is present at picomolar concentrations (10⁻¹⁰ -10 ~10 -12 It exists only in M). See Alexander WS. Int Rev Immunol. 1998, 16: 651-682; Gasson JC. Blood. 1991, 77: 1131-1145; Shannon MF et al. Crit Rev Immunol. 1997, 17: 301-323; Barreda DR et al. Dev Comp Immunol. 2004, 28: 509-554; and Metcalf D. Immunol Cell Biology. 1987, 65: 35-43.

[0006] Human GM-CSF (hGM-CSF) is synthesized as a precursor protein consisting of 144 amino acid residues, including a 17-amino acid signal peptide. Processing of this precursor protein yields a mature protein of 127 amino acids with a predicted molecular weight of 14.4 kDa. This mature protein possesses two disulfide bonds and electrophores in a broadband range of 15–30 kDa due to glycosylation and sialylation. The glycosylation pattern of GM-CSF has been observed to affect its activity, receptor binding, immunogenicity, and half-life. See Lee F. et al. Proc Natl Acad Sci USA Biochem. 1985.82:360-4364; Miyatake S. et al. EMBO J. 1985.4:2561-2568; Cebon J et al. J Biol. Chem. 1991.265,4483-4491; and Zhang Q et al. Proc. Natl. Acad. Sci. 2014..2885-2890.

[0007] Recombinant human granulocyte-macrophage colony-stimulating factor (rhu GM-CSF) is approved by the FDA for use in combination with chemotherapy agents in the treatment of neutropenia, hematological disorders, and malignancies such as leukemia. Clinically, GM-CSF used to treat neutropenia and aplastic anemia after chemotherapy significantly reduces the risk of infection associated with bone marrow transplantation. The usefulness of GM-CSF in the treatment of myeloid leukemia and as a vaccine adjuvant is also well established. See Dorr RT. Clin Therapeutics. 1993. 15(1):19-29; Armitage JO. Blood 1998, 92:4491-4508; Kovacic JC et al. J Mol Cell Cardiol. 2007, 42:19-33; Jacobs PP et al. Microbial Cell Factories 2010, 9:93.

[0008] There are five types of heterologous protein production platforms, including bacterial, yeast, plant, insect, and mammalian cells. However, more than 50% of currently marketed biopharmaceuticals are produced using mammalian cell lines. This is partly because the remaining four types cannot modify glycoproteins with human-like oligosaccharides. This is important because protein-binding glycans affect circulating half-life, tissue distribution, biological activity, and immunogenicity. The GM-CSF expression systems described above affect the pharmacokinetic properties, biological activity, and clinical toxicity of GM-CSF. Clinically, GM-CSF is produced using Chinese hamster ovary cells (CHO-GM, reglamostim), Escherichia coli (E. coli-GM, morglamostim), or yeast (yeast GM, salglamostim). See Dorr RT. Clin Therapeutics. 1993. 15(1):19-29; Walsh G. Nat Biotechnol. 2006, 24:769-776; Jacobs PP et al. Nat Protoc. 2009, 4:58-70; Jacobs PP et al. Microbial Cell Factories 2010, 9:93; Walsh G. Nat Biotechnol. 2018, 36(12):1136-1145.

[0009] Basic nutritional requirements for all microorganisms include water and oxygen, as well as carbon, nitrogen, vitamins, and mineral elements. Mineral requirements for yeast vary depending on the specific strain and culture growth conditions. Generally, yeast has two types of mineral requirements: macronutrients, which are needed in large quantities, and trace elements, which are needed in small quantities. Examples of trace elements include iron, copper, zinc, manganese, molybdenum, cobalt, boron, and others. These trace elements are essential for yeast growth and play a crucial role in cellular metabolism, primarily because they are required as cofactors for numerous enzymes. In the Salgramostim cell growth step of the manufacturing process (flask shaking, seed fermentation), the host organism's mineral requirements are met by adding a trace element solution to the culture medium. However, trace elements are not added during production fermentation; instead, a complex blend of two protein hydrolysates (Bacto-Peptone, yeast extract) is used to meet all mineral requirements.

[0010] Bacto-Peptone and yeast extract are used in the salglamostim production process as a source of complex organic nitrogen, inorganic nitrogen, vitamins, trace elements, and free amino acids for yeast culture during production fermentation, thereby promoting cell proliferation and the expression and secretion of salglamostim. The heterogeneous nature of these materials and the resulting lot-to-lot variability have been shown to significantly affect yeast culture performance, productivity, and product quality. Consequently, growth rate and productivity can be greatly influenced by fluctuations in unknown minerals supplied to the culture via the complex medium.

[0011] To improve yield consistency and efficiency, there remains a need to reduce variations in micronutrients during the production process of rhu GM-CSF. [Overview of the Initiative]

[0012] Therefore, the present invention relates in part to copper, an essential trace element in yeast, as a major limiting component in culture media that affects productivity. For example, this disclosure demonstrates, among other things, that copper (Cu) is a limiting trace element in Bacto Peptone and yeast extract. Addition of additional copper to the above culture media improved low-productivity lots and significantly increased yields.

[0013] In some embodiments, a method for producing recombinant proteins is provided, comprising adding a trace element, copper, to a culture medium containing host cells such as yeast. The host cells contain nucleic acid molecules encoding the recombinant protein, for example, rhu GM-CSF, and have the ability to produce this protein during fermentation, and the trace element is added exogenously to the culture medium to supplement the amount of trace elements in the culture medium.

[0014] In some embodiments, a manufacturing method is also provided that uses a nucleic acid molecule encoding the recombinant human GM-CSF (e.g., a codon-optimized sequence). In some embodiments, a manufacturing method is also provided that uses a non-human host cell expressing the nucleic acid molecule encoding the recombinant human GM-CSF (e.g., yeast cells, e.g., non-methylotrope yeast cells, e.g., Saccharomyces cerevisiae). In some embodiments, a pharmaceutical composition is also provided that comprises the recombinant human GM-CSF produced by the manufacturing method and a pharmaceutically acceptable excipient or carrier.

[0015] In some embodiments, there is a method for treating a patient or subject who is currently receiving or has previously received cancer treatment, or who has received or has previously received a bone marrow transplant, and / or who has been acutely exposed to a bone marrow suppression dose of radiation, the method comprising administering to the patient a therapeutically effective amount of a pharmaceutical composition produced by the manufacturing method described herein.

[0016] In some embodiments, a method for treating a viral infection, for example, an infection caused by a coronavirus, for example, not limited to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is provided, comprising administering an effective amount of a pharmaceutical composition produced by the manufacturing method described herein, or a method for treating or preventing a viral infection in a subject requiring such a method, comprising preparing plasma derived from a donor subject who has recovered from an infection caused by the viral infection, for example, not limited to a coronavirus, for example, not limited to SARS-CoV-2 (provided that the plasma contains IgG, IgM, and / or IgA antibodies against the virus causing the infection, and the donor subject has been treated with recombinant human GM-CSF protein produced by the manufacturing method described herein to stimulate the production of the antibodies), and administering the plasma to a subject requiring such a method.

[0017] In some embodiments, a method for producing a recombinant that produces a composition containing recombinant human GM-CSF is provided, comprising: (a) adding an exogenous trace element, copper, to a culture medium containing host cells such as yeast (provided that this trace element is added exogenously to the culture medium to supplement the amount of trace elements in the culture medium and reach a target concentration range); (b) transfecting yeast cells with a nucleic acid encoding recombinant human GM-CSF that contains an amino acid sequence that is at least about 97% identical, at least about 98%, or at least about 99% identical to the amino acid sequence of SEQ ID NO: 1 and / or SEQ ID NO: 2, or has the amino acid sequence of SEQ ID NO: 1 and / or SEQ ID NO: 2; and (c) host cells having the ability to produce this protein with high efficacy and consistency during fermentation.

[0018] In some embodiments, the present invention relates to a method for improving the production of a physiologically active substance such as recombinant human GM-CSF, comprising adding exogenous copper to a culture medium for producing the physiologically active substance, which can be obtained by culturing animal cells or cell lines capable of producing the physiologically active substance in the culture medium.

[0019] More specifically, the present invention relates, in some embodiments, to a method for producing a bioactive substance, comprising culturing animal cells (e.g., yeast cells) or cell lines (e.g., CHO cells) capable of producing the bioactive substance in a culture medium containing exogenous copper to produce the bioactive substance, and isolating the bioactive substance from the culture medium.

[0020] A patent or application file must include at least one drawing created in color. A copy of this patent or patent application publication containing the color drawing is available from the Patent Office upon application and payment of the required fees. [Brief explanation of the drawing]

[0021] [Figure 1A] This graph shows the effect of various trace elements on the amount of dissolved oxygen after addition to yeast cell cultures. It displays the dissolved oxygen profiles after individual screening of each trace element. The bottom curve represents "copper." [Figure 1B] This graph shows the effect of adding exogenous copper to yeast cell cultures on dissolved oxygen levels, compared to a scaled-down commercial process. It also shows dissolved oxygen profiles comparing simultaneous fermentation with a scaled-down commercial process and copper addition. At 15.0 hours, the upper curve represents the "scaled-down commercial process," and the lower curve represents the "copper addition" method. [Figure 2A]A graph showing the effect of adding various trace elements on the wet cell weight of yeast after addition to yeast cell cultures. A cell wet weight profile showing a comparison of individually screened trace elements is presented. At the 20-hour point, the topmost curve is "copper", followed by "zinc", "molybdate", "manganese", "iron", and "boron" in descending order from top to bottom. [Figure 2B] A graph showing the effect of adding exogenous copper on the wet cell weight of yeast after addition to yeast cell cultures compared to a scaled-down commercial process. A bar graph of cell wet weight showing a comparison of the fermentation conducted simultaneously with the scaled-down commercial process and copper addition is presented. [Figure 3] A graph showing the titer of recombinant human GM-CSF obtained during simultaneous fermentation with and without the addition of exogenous copper. A bar graph of various titers showing a comparison of the fermentation conducted simultaneously with the scaled-down commercial process and copper addition is presented. [Figure 4] A figure showing the results of an SDS-PAGE - silver staining (T-0002) assay for evaluating the impurities of a CuSO4 batch (CuSO4 PV) in BDS compared to commercial BDS batches 6 - 8. Each gel contains a reference standard sample, a molecular weight marker, as well as reduced and non-reduced samples. The identities of the samples are as follows. BDS 6: reduced reference standard sample (lane 2), reduced BDS 6 (lane 4), non-reduced reference standard sample (lane 7), and non-reduced BDS 6 (lane 9). BDS 7: reduced reference standard sample (lane 2), reduced BDS 7 (lane 5), non-reduced reference standard sample (lane 7), and non-reduced BDS 7 (lane 10). BDS 8: reduced reference standard sample (lane 2), reduced BDS 8 (lane 3), non-reduced reference standard sample (lane 7), and non-reduced BDS 8 (lane 8). CuSO4 PV: reduced reference standard sample (lane 2), reduced CuSO4 PV (lane 3), non-reduced reference standard sample (lane 7), non-reduced CuSO4 PV non-reduced (lane 8). [Figure 5]This figure shows the results of a concentration measurement test (T-0013) to evaluate the protein purity level of salglamostim in CuSO4 batches (CuSO4 PV) in BDS compared with commercial BDS batches 6-8. Each gel contains a reference standard sample lane (lane 4), a thermal molecular weight marker (lane 2), and a commercial BDS sample or PV sample (lane 6). [Figure 6] This figure shows the results of isoelectric focusing (T-0114) used to measure the identity of salglamostim in CuSO4 batches (CuSO4 PV) in BDS compared with commercial BDS batches 6-8. Each gel contains a GE Healthcare pI marker (lane 2), a reference standard sample (lane 4), and a commercial BDS or PV sample (lane 6). [Figure 7] This graph shows the ELISA results for CuSO4 PV batches (CuSO4 PV) across all historical batches, illustrating the removal of residual process components (RPCs) throughout the purification process. The dotted line represents the average value of all historical commercial data, and the solid line represents CuSO4 batches (CuSO4 PV) at the BDS level. Commercial BDS batches 6-8 are shown only at the BDS level. The results for all historical commercial batches, CuSO4 PV, and BDS 6-8 are very similar and overlap. [Figure 8] This figure shows the peak separation of RP-HPLC chromatography, demonstrating C-terminal analysis performed using trypsin-based peptide mapping (TCPK-trypsin). Peaks A (amino acids 86-107), B (amino acids 108-111), and C (amino acids 112-127) are shown for CuSO4 PV and commercial BDS 6-8, respectively. [Figure 9] This figure shows peptide maps mediated by Glu-C at low pH, indicating disulfide crosslinking. These chromatograms show peaks 11 and 12, which contain disulfide peptide fragments confirmed by mass spectrometry. This figure shows CuSO4 batches (CuSO4 PV) in BDS and commercial BDS batches 6-8. [Figure 10]This figure shows a chromatogram of the peptide map (78.5–82.5 min) with Glu-C at low pH, including peptides G3–4 and deamidated fragments. This result indicates the proportion (site occupancy) of the total N-linked glycosylation at position 27. This figure shows the CuSO4 batch (CuSO4 PV) in BDS and commercial BDS batches 6–8. [Figure 11] This figure shows a chromatogram of a peptide map using Glu C without α-mannosidase, containing glycosylated G1 peptide, unglycosylated Ala 3, and unglycosylated Ala 1 peptide fragments. The size (site occupancy) of all O-linked glycosylated chains was measured as a percentage, representing the ratio of the total area of ​​O-linked glycoform peaks to the area of ​​unmodified peaks. This figure shows CuSO4 batch (CuSO4 PV) in BDS and commercial BDS batches 6-8. [Figure 12] This figure shows a chromatogram of the peptide map with Glu C at neutral pH, including G9 and oxidized fragments. Oxidation at methionine 79 was measured by mass spectrometry. This figure shows CuSO4 batches (CuSO4 PV) in BDS and commercial BDS batches 6-8. [Figure 13] The emission fluorescence spectra from 305 nm to 405 nm, with the blank subtracted, are shown at excitation = 295 nm. The graph of this spectrum shows the equivalence of the thermal stability of the protein structure when measured at 10 to 90°C. This figure shows graphs for CuSO4 batch (CuSO4 PV) in BDS and commercial BDS batches 6 to 8. The curves start at the top of Figure 13 and show measurements from approximately 10°C to 18°C ​​(purple curve), approximately 20°C to 32°C (blue curve), approximately 34°C to 46°C (green curve), approximately 48°C to 52°C (yellow curve), approximately 54°C to 58°C (orange curve), approximately 60°C to 80°C (red curve), and approximately 82°C to 90°C (brown curve), ending at the bottom of Figure 13. [Figure 14]To demonstrate lot-to-lot equivalence of protein structures in solution, the spectral mass center of the emission spectrum at 305–405 nm is shown. This figure shows graphs of CuSO4 batch (CuSO4 PV) in BDS and commercial BDS batches 6–8. [Figure 15] A graph comparing circular dichroism (CD) spectra (5-10°C and 90°C) is shown. CD scanning and thermal unfolding data (Tm and T start) demonstrate equivalence among all four BDS (CuSO4 PV and commercial BDS 6-8) lots tested. The red line indicates absorption at 90°C, and the blue line indicates absorption at 10°C. [Figure 16] The graphs show intact or complete MALDI-MS mass spectral analyses for 12–19 kDa samples. These graphs represent the observed spectral masses for all four BDS (CuSO4 PV and commercial BDS 6–8) lots tested. All MALDI-MS imaging was performed at the Fred Hutchinson Cancer Research Center Proteomic Facility using an Applied Biosystems 4800 MALDI-TOF / TOF. Samples were diluted 10-fold with synapic acid, spotted onto MALDI plates, and MS was acquired for 15 minutes per sample from 2–19 kDa. [Figure 17] The MALDI-MS mass spectral analyses of salglamostim at 14–19 kDa are shown. These graphs show the spectral masses observed for all four BDS (CuSO4 PV and commercial BDS 6–8) lots tested. [Figure 18] The MALDI-MS mass spectral analysis of salglamostim at 16–19 kDa is shown. These graphs show the spectral masses observed for all four BDS (CuSO4 PV and commercial BDS 6–8) lots tested. [Modes for carrying out the invention]

[0022] This invention is partly based on the discovery that the yield of recombinant human GM-CSF (rhu GM-CSF) is increased by exogenously adding copper (Cu), a single micronutrient, during production. Furthermore, this invention is based on the discovery that this improvement in production efficiency did not affect the quality of the rhu GM-CSF produced.

[0023] In some embodiments, the present invention provides an improved method for producing physiologically active substances such as rhu GM-CSF, which involves adding exogenous copper to a culture medium used for the production of the physiologically active substances by cultured animal cells (such as yeast cells) or cell lines (such as CHO cells).

[0024] Manufacturing method Embodiments provided herein offer a method for achieving consistent and efficient production of recombinant glycoproteins such as rhu GM-CSF, comprising increasing the concentration of copper in a cell culture containing host cells producing the recombinant glycoprotein of interest to reach a target concentration range.

[0025] Embodiments provided herein are a method for improving a cell culture medium for the production of recombinant rhu GM-CSF, comprising: (i) measuring the amount of copper in the cell culture medium or components used to manufacture the cell culture medium; and (ii) adjusting the concentration of copper in the cell culture medium to reach an amount of copper within a predetermined target range, wherein the target range is sufficient to produce the recombinant glycoprotein of interest with improved consistency and yield.

[0026] Embodiments provided herein provide a method for improving the production of physiologically active recombinant glycoproteins such as rhu GM-CSF, comprising: (i) measuring the amount of copper in a yeast cell culture; and (ii) if the amount of copper is below a target range, adding copper to the yeast cell culture to bring the amount of copper to a target range.

[0027] In some embodiments, a method for producing a recombinant that produces a composition containing recombinant human GM-CSF is provided, comprising: (a) obtaining yeast cells or an extract thereof transfected with a nucleic acid encoding recombinant human GM-CSF having an amino acid sequence having at least about 90%, at least about 93%, at least about 95%, at least about 97%, at least about 98%, or at least about 99% identity with SEQ ID NO: 1 or SEQ ID NO: 2; (b) purifying the GM-CSF from the transfected yeast cells in the absence of organic solvents using one or more HPLC columns; and (c) recovering the purified GM-CSF, wherein the purified GM-CSF substantially does not contain excessively glycosylated, for example, excessively mannosylated GM-CSF forms.

[0028] In some embodiments, the yeast is S. cerevisiae.

[0029] In some embodiments, the method further includes formulating purified GM-CSF for injection, such as subcutaneous or intravenous injection.

[0030] Culture medium In some embodiments, the culture medium of the present invention is not particularly limited as long as it can maintain the survival and proliferation of animal cells (such as yeast cells) or cell lines (such as CHO cells). Examples include a culture medium comprising a carbon source that can be assimilated by animal cells, a nitrogen source that can be digested by animal cells, and vitamins and / or mineral elements. In some embodiments, the culture medium comprises Bacto-Peptone and / or yeast extract.

[0031] In some embodiments, the mineral elements of the present invention include macroelements and trace elements. Examples of such macroelements include carbon, hydrogen, oxygen, and nitrogen. Examples of trace elements include copper, iron, zinc, manganese, molybdenum, cobalt, and boron.

[0032] In some embodiments, additional exogenous trace mineral elements, such as copper, are added to the culture medium. Addition to the cell culture medium, as in the present invention, allows for control of manufacturing efficiency and productivity.

[0033] While we do not wish to be bound by theory, the nutritional requirements of yeast that may affect growth rate and viability (Due C et al., PLOS One, 12(9):1-22; Broach JR Genetics. 192(1)73-105, 2012; Gadd GM, FEMS Microbial Lett. 79:197-203, 1992).

[0034] copper In some embodiments, copper may be added to the cell culture medium in the form of copper sulfate, or cupric sulfate. Depending on the specific medium, copper is added to the cell culture medium in an amount of about 0.5 μM to about 100 μM (optionally about 0.5 μM to about 80 μM, or optionally about 1 μM to about 20 μM).

[0035] In some embodiments, copper may be added to the cell culture in the form of copper sulfate (cupric), copper oxide, copper chloride, copper iodide, copper sulfide, copper acetylide, copper bromide, copper fluoride, copper hydroxide, copper hydride, copper nitrate, copper phosphide, copper acetate, copper carbonate, copper chlorate, or copper phosphate.

[0036] Therefore, in some embodiments, this information can provide those skilled in the art with information regarding acceptable variations of the copper salt.

[0037] fermentation In some embodiments, the present invention provides a method comprising fermentation for producing protein products.

[0038] In various embodiments, the production of the recombinant protein, e.g., rhu GM-CSF by genetic engineering, may consist of a series of 10 or up to 10 different unit operations. In some embodiments, the production fermentation process of the recombinant protein (e.g., salglamostim) generates, harvests, and recovers rhu GM-CSF. During the upstream production process, four major GM-CSF species are present in the partially purified fermenter culture medium, including an over-glycosylated isoform, N- and N-+O-glycosylated isoform, O-glycosylated isoform, and a non-glycosylated (approximately 15 kDa, peak 4) species.

[0039] In various embodiments, the fermentation process comprises three stages: 1.5L flask shaking, 15L seed fermentation, and 100L production fermentation. The 1.5L flask shaking step allows the preliminary yeast culture in the working cell bank vial to grow to a sufficient volume and density for inoculation into the 15L seed fermentation process. The 15L seed fermentation process allows the culture to grow further to a sufficient volume and density for inoculation into the 100L production fermentation. The 100L production fermentation is a fed-batch process that increases biomass and promotes the expression and secretion of recombinant proteins, such as rhu GM-CSF, which are then recovered and purified. In some embodiments, at the end of the 100L production fermentation process, the fermented cultures are combined and recovered by microfiltration and ultrafiltration.

[0040] Isolation In some embodiments, the present invention provides a method comprising an isolation method for obtaining a protein product. In some embodiments, recombinant proteins, such as genetically engineered rhu GM-CSF, are isolated or purified by gel filtration chromatography, ion exchange chromatography, affinity chromatography, or high-performance liquid chromatography based on properties such as solubility, size, charge, and specific binding affinity.

[0041] In some embodiments, the purification or isolation of the recombinant protein, for example, genetically engineered rhu GM-CSF, is carried out in a downstream process consisting of three reverse-phase high-performance liquid chromatography (RP-HPLC) operations, one low-pressure cation exchange chromatography operation, and a final filtration operation. In some embodiments, the purification or isolation step may include a C4 capture process, a C4 purification process, and a C18 purification process.

[0042] Composition of GM-CSF In one embodiment, the genetically engineered rhu GM-CSF produced using the present invention, specifically the addition of exogenous copper, is identical to recombinant human GM-CSF (rhu GM-CSF) such as salglamostim (LEUKINE). Salglamostim is a biosynthetically synthesized, yeast-derived recombinant human GM-CSF having a single 127-amino acid glycoprotein, distinct from endogenous human GM-CSF in that it has leucine instead of arginine at position 23. Other natural and synthetic GM-CSFs, and their derivatives, possessing the biological activity of natural human GM-CSF may also be similarly useful in the implementation of the present invention.

[0043] While we do not wish to be bound by theory, the degree of glycosylation of biosynthesized GM-CSF appears to affect its half-life, distribution, and excretion (Lieschke and Burgess, N.Engl.J.Med.327:28-35,1992; Dorr, RT, Clin.Ther 15:19-29,1993; Horgaard et al., Eur.J.Hematol.50:32-36,1993).

[0044] In one embodiment, a recombinant human GM-CSF is provided that has at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity, or 100% identity, with SEQ ID NO: 1 or SEQ ID NO: 2.

[0045] In some embodiments, the GM-CSF is one of morglamostim, salglamostim, and reglamostim.

[0046] While we do not wish to be constrained by theory, the core of hGM-CSF is composed of four angled, packed helices. Analysis of the crystal structure and mutagenicity of recombinant human GM-CSF (Rozwarski DA et al., Proteins 26:304-13, 1996) revealed that, in addition to the nonpolar side chains in the core of the protein, 10 embedded hydrogen-bonding residues are involved in intramolecular hydrogen bonding with the main chain atoms, which is more conserved than residues hydrogen-bonding to other side chain atoms; 24 solvation sites were observed at equivalent positions in two molecules within the asymmetric unit, with the strongest solvation sites among them located in the fissures between secondary structure elements. Two surface clusters of hydrophobic side chains are located near the expected receptor-binding region.

[0047] Furthermore, in some embodiments, those skilled in the art may refer to UniProtKB entry P04141 for structural information that conveys information about the identity of a variant.

[0048] The N-terminal helix of hGM-CSF governs its high-affinity binding to its receptor (Shanafelt AB et al., EMBO J 10:4105-12, 1991). Transduction of the biological effects of GM-CSF requires interaction with at least two cell surface receptor components (one of which is shared with the cytokine IL-5). In the above study, receptor-binding determinants in GM-CSF were identified by determining the location of intrinsic receptor-binding domains on a series of human-mouse hybrid GM-CSF cytokines. The interaction between GM-CSF and its high-affinity receptor complex's shared subunit was governed by only a small portion of the peptide chain. The presence of only a few key residues in the N-terminal α-helix of GM-CSF was sufficient to confer specificity to the above interaction.

[0049] In some embodiments, the genetically modified GM-CSF used in the implementation of the present invention may be any pharmaceutically safe and effective GM-CSF, or any derivative thereof having the biological activity of GM-CSF.

[0050] In some embodiments, the rhu GM-CSF molecule comprises multiple molecular forms similar to salglamostim. In some embodiments, these molecular forms are selected from a non-glycosylated form, an O-glycosylated form, an N-glycosylated form, and an N+O glycosylated form. Furthermore, in some embodiments, the recombinant human GM-CSF substantially does not contain excessively glycosylated forms, such as excessively mannosylated forms.

[0051] In some embodiments, the rhu GM-CSF molecule comprises two or more species (e.g., glycoforms). In some embodiments, the molecular weight of any of the above species does not exceed approximately 20 kDa.

[0052] Functional characteristics of recombinant GM-CSF In some embodiments, the recombinant human (rhu)GM-CSF molecule produced with the addition of exogenous copper is functionally similar to wild-type human GM-CSF and / or salglamostim produced without the addition of exogenous copper (for example, differences in one or more functional parameters of about 50% or less, or about 40% or less, or about 30% or less, or about 20% or less, or about 10% or less, or about 5% or less, or differences in the functional parameters assayed of about 1 / 5 or less, or about 1 / 4 or less, or about 1 / 3 or less, or about 1 / 2 or less). In some embodiments, the above functional parameters of GM-CSF can be detected by assays known in the art, such as proliferation assays using cells such as TF-1 cell lines and primary myeloid cells, biochemical assays such as iLite® GM-CSF (luciferase under the control of the GM-CSF promoter), cell viability assays such as myeloid cell viability assays, cell differentiation assays, and co-culture experiments.

[0053] In some embodiments, the rhu GM-CSF molecule, manufactured with the addition of exogenous copper, can bind to and / or activate granulocyte-macrophage colony-stimulating factor receptors (GM-CSF-R-alpha or CSF2R). In some embodiments, the rhu GM-CSF molecule, manufactured with the addition of exogenous copper, can bind to and / or activate granulocyte-macrophage colony-stimulating factor receptors (GM-CSF-R-alpha or CSF2R) with affinity, efficacy, and / or bioactivity (e.g., differences in one or more functional parameters of about 50% or less, or about 40% or less, or about 30% or less, or about 20% or less, or about 10% or less, or about 5% or less, or about 1 / 5 or less, or about 1 / 4 or less, or about 1 / 3 or less, or about 1 / 2 or less) equivalent to wild-type human GM-CSF and / or salglamostim manufactured without the addition of exogenous copper. Assays for binding and activation by GM-CSF are known in the art. Non-limiting examples of such assays include, for example, radioactive ligand assays or non-radioactive ligand assays (e.g., immunoprecipitation (IP), enzyme-linked immunosorbent assay (ELISA), Western blotting, fluorescence polarization (FP), fluorescence resonance energy transfer (FRET), surface plasmon resonance (SPR), and radioimmunoassay (RIA)). Binding kinetics can also be evaluated by standard assays known in the art, such as Biacore analysis. Whole-cell ligand binding assays and cell-free assay systems using soluble GM-CSF receptor alpha (sGMRa) may also be used. Other types of assays that can be used include receptor binding assays, or saturated binding assays, or competitive binding assays using radioactive iodized GM-CSF, as well as cell proliferation assays.

[0054] In some embodiments, the rhu GM-CSF molecule can be assayed using one or more cell-based activity bioassays, for example, using a GM-CSF-dependent human cell line proliferation assay using TF-1, M-07e, HU-3, M-MOK, MB-02, GM / SO, F-36P, GF-D8, ELF-153, AML-193, MUTZ-3, OCI-AML5, OCI-AML6, OCI-AML1, SKNO-1, UCSD-AML1, and UT-7.

[0055] In some embodiments, the potency of the rhu GM-CSF molecule is measured using a bioassay with TF-1 cells, a human erythroleukemia cell line that proliferates in response to GM-CSF. Details of this assay are known in the art. For example, a reference standard, control, and test sample are serially diluted in assay medium in n=3 and added to three separate 96-well plates. Then, TF-1 cells in suspension are added, and the mixture is incubated at 37°C for 69.5–72 hours. After adding a fluorescent dye (e.g., ALAMARBLUE), the plates are incubated at 37°C for 6.6–8 hours. Next, the proliferation of TF-1 cells is measured using a fluorescence microplate reader.

[0056] In some embodiments, GM-CSF-R-alpha, which binds and / or activates, is expressed on the surface of cells. In some embodiments, the cells are hematopoietic progenitor cells. In some embodiments, the hematopoietic progenitor cells are immune cells. In some embodiments, the hematopoietic progenitor cells are irradiated.

[0057] In some embodiments, the immunogenicity of the rhu GM-CSF molecule with the present substitutions and / or deletions is equivalent to that of wild-type human GM-CSF and / or salglamostim (e.g., differences in one or more functional parameters of about 50% or less, or about 40% or less, or about 30% or less, or about 20% or less, or about 10% or less, or about 5% or less, or about 1 / 5 or less, or about 1 / 4 or less, or about 1 / 3 or less, or about 1 / 2 or less). In some embodiments, immunogenicity is assayed using methods known in the art. Non-limiting examples include, for example, detection of one or more anti-GM-CSF binding antibodies, which are evaluated by screening assays such as direct ELISA, indirect ELISA, or bridging ELISA, electrochemiluminescence, bead-based chemiluminescence assays, radioimmunoprecipitation assays, surface plasma resonance, and biolayer interferometry, as well as cell-based luciferase reporter gene neutralizing antibody assays.

[0058] In some embodiments, recombinant human GM-CSF is soluble.

[0059] Nucleic acids and host cells In some embodiments, nucleic acid molecules encoding recombinant human GM-CSF described herein are provided. In some embodiments, the nucleic acid molecules have a codon-optimized sequence.

[0060] In some embodiments, non-human host cells expressing the nucleic acid molecules described herein are provided. In some embodiments, the host cells are yeast cells.

[0061] In some embodiments, the yeast cells are non-methylotrope yeast cells. In some embodiments, the host cells are Saccharomyces cerevisiae cells.

[0062] In some embodiments, the host cells are mammalian cells. In some embodiments, the host cells are CHO (Chinese hamster ovary) cells, NSO (mouse myeloma) cells, BHK (baby hamster kidney) cells, Sp2 / 0 (mouse myeloma) cells, human retinal cells, HUVEC cells, HMVEC cells, COS-1 cells, COS-7 cells, HeLa cells, HepG-2 cells, HL-60 cells, IM-9 cells, Jurkat cells, MCF-7 cells, or T98G cells, etc.

[0063] Pharmaceutical compositions and preparations In some embodiments, pharmaceutical compositions comprising recombinant human GM-CSF and pharmaceutically acceptable excipients or carriers as described herein are provided.

[0064] Any of the pharmaceutical compositions described herein can be administered to a subject as a component of a composition comprising a pharmaceutically acceptable carrier or vehicle. Such a composition may optionally contain suitable amounts of pharmaceutically acceptable excipients to provide a form for appropriate administration.

[0065] In various embodiments, the pharmaceutical excipient may be a liquid, such as water and oils of petroleum, animal, plant, or synthetic origin, including peanut oil, soybean oil, mineral oil, sesame oil, etc. The pharmaceutical excipient may also be, for example, physiological saline, gum arabic, gelatin, starch paste, talc, keratin, colloidal silica, urea, etc. In addition, auxiliary agents, stabilizers, thickeners, lubricants, and colorants may be used. In one embodiment, the pharmaceutically acceptable excipient is sterile when administered to a subject. Water is a useful excipient when administering any of the drugs described herein intravenously. Physiological saline and aqueous solutions of dextrose and glycerol can also be used as liquid excipients, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, and ethanol. Any of the agents described herein may optionally contain small amounts of wetting agents, emulsifiers, or pH buffers. Other examples of suitable pharmaceutical excipients are described in Remington's Pharmaceutical Sciences 1447-1676 (Alfonso R. Gennaro eds., 19th ed. 1995), which is incorporated herein by reference.

[0066] The present invention includes, in several embodiments, pharmaceutical compositions (and / or further therapeutic agents) described herein in various formulations. Any of the pharmaceutical compositions (and / or further therapeutic agents) of the present invention described herein may take the form of a solution, suspension, emulsion, drop, tablet, pill, pellet, capsule, liquid capsule, gelatin capsule, powder, sustained-release formulation, suppository, emulsion, aerosol, spray, suspension, lyophilized powder, freeze suspension, dry powder, or any other form suitable for use. In one embodiment, the composition is in the form of a capsule. In another embodiment, the composition is in the form of a tablet. In yet another embodiment, the pharmaceutical composition is formulated in the form of a soft gel capsule. In yet another embodiment, the pharmaceutical composition is formulated in the form of a gelatin capsule. In yet another embodiment, the pharmaceutical composition is formulated as a liquid.

[0067] If necessary, the pharmaceutical composition (and / or further therapeutic agent) of the present invention may contain a solubilizer. The agent may also be delivered using a suitable vehicle or delivery device known in the art. The combination therapeutic agents outlined herein may be delivered simultaneously by a single delivery vehicle or delivery device.

[0068] Formulations comprising the pharmaceutical composition (and / or further therapeutic agents) of the present invention may, in some embodiments, be provided in the form of convenient unit dosage forms and can be formulated by any method well known in the field of pharmaceuticals. Such methods generally include the step of mixing the therapeutic agent with a carrier, the carrier comprising one or more auxiliary components. Generally, the formulation is formulated by uniformly and tightly mixing the therapeutic agent with a liquid carrier, a micronized solid carrier, or both, and then excipienting the product into a desired formulation (e.g., wet or dry granulation, powder formulation, etc., followed by tableting using conventional methods known in the art).

[0069] In various embodiments, any of the pharmaceutical compositions (and / or further therapeutic agents) described herein are formulated according to conventional procedures as compositions suitable for the mode of administration described herein.

[0070] Routes of administration include, for example, oral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, sublingual, intranasal, intracerebral, vaginal, transdermal, rectal, inhalation, or local administration. Administration may be local or systemic. In some embodiments, the administration is performed orally. In other embodiments, the administration is performed by parenteral injection. The mode of administration is at the discretion of the physician and in part depends on the site of the disease. In most cases, administration results in the release of one of the drugs described herein into the bloodstream.

[0071] In certain embodiments, the GM-CSF (and / or further therapeutic agents) are administered via an intravenous route.

[0072] In one embodiment, the pharmaceutical composition (and / or further therapeutic agent) described herein is formulated according to conventional procedures as a composition suitable for oral administration. The composition for oral delivery may be, for example, in the form of tablets, lozenges, aqueous or oily suspensions, granules, powders, emulsions, capsules, syrups, or elixirs. The composition for oral administration may contain one or more additives to provide a pharmaceutically palatable formulation, such as sweeteners such as fructose, aspartame, or saccharin; flavorings such as peppermint, wintergreen oil, or cherry; colorings; and preservatives. Furthermore, in the form of tablets or pills, the composition may be coated to delay disintegration and absorption in the gastrointestinal tract, thereby providing a long-lasting effect. A selectively permeable membrane surrounding an osmotic activator driving any of the pharmaceutical compositions (and / or further therapeutic agents) described herein is also suitable for oral administration compositions. In these latter platforms, fluids from the environment surrounding the capsule are absorbed by the driving compound, which swells and releases the drug or drug composition through the opening. These delivery platforms can provide an essentially zero-order delivery profile, in contrast to the steeply rising profile of immediate-release formulations. Time-delaying substances such as glycerol monostearate and glycerol stearate may also be useful. Oral compositions may contain standard excipients such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, and magnesium carbonate. In one embodiment, the excipients are pharmaceutical-grade excipients. In addition to the active compound, suspensions may contain suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol, and sorbitan esters, microcrystalline cellulose, aluminum methhydrooxide, bentonite, agar, tragacanth, and mixtures thereof.

[0073] Suitable dosage forms for parenteral administration (e.g., intravenous, intramuscular, intraperitoneal, subcutaneous, and intra-articular injection and infusion) include, for example, solutions, suspensions, dispersions, and emulsions. These may be manufactured in the form of sterile solid compositions (e.g., lyophilized compositions), which can be dissolved or suspended in a sterile injection medium immediately before use. The compositions may contain, for example, suspensions or dispersants known in the art. Suitable formulation components for parenteral administration include sterile diluents such as water for injection, physiological saline, non-volatile oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antimicrobial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as EDTA; buffering agents such as acetates, citrates, or phosphates; and additives for adjusting tonicity such as sodium chloride or dextrose.

[0074] For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). The carrier must be stable under manufacturing and storage conditions and must be stored in a way that prevents spoilage by microorganisms. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof.

[0075] Any of the pharmaceutical compositions (and / or further therapeutic agents) of the present invention described herein may be administered by controlled-release or sustained-release means, or by delivery devices well known to those skilled in the art. Examples include, but are not limited to, those described in U.S. Patents 3,845,770, 3,916,899, 3,536,809, 3,598,123, 4,008,719, 5,674,533, 5,059,595, 5,591,767, 5,120,548, 5,073,543, 5,639,476, 5,354,556, and 5,733,556, each of which is incorporated herein by reference in its entirety. For example, such dosage forms using hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes, microspheres, or combinations thereof may be useful in providing controlled or sustained release of one or more active ingredients and in providing desirable release profiles in various proportions. Suitable controlled-release or sustained-release formulations known to those skilled in the art, including those described herein, can be readily selected for use with the active ingredients of the drugs described herein. Accordingly, the present invention provides, in some embodiments, single unit dosage forms suitable for oral administration, such as, but not limited to, tablets, capsules, gel caps, and caplets, which are adapted for controlled or sustained release.

[0076] The controlled or sustained release of the active ingredient can be stimulated by a variety of conditions, including, but not limited to, changes in pH, changes in temperature, stimulation with light of an appropriate wavelength, enzyme concentration or availability, water concentration or availability, or other physiological conditions or compounds.

[0077] In another embodiment, the controlled-release system can be positioned close to the target area to be treated, thus requiring only a fraction of the systemic dose (see, for example, Goodson, Medical Applications of Controlled Release, supra, vol.2, pp.115-138 (1984)). Other controlled-release systems may be used, as discussed in the review by Langer, 1990, Science 249:1527-1533.

[0078] Pharmaceutical preparations are preferably sterile. Sterilization can be achieved, for example, by filtration through a sterile filter membrane. If the above composition is freeze-dried, filter sterilization can be performed before or after freeze-drying and reconstitution.

[0079] Pharmacopoeia-acceptable salts and excipients The compositions described herein may have a sufficiently basic functional group that can react with an inorganic or organic acid to form a pharmaceutically acceptable salt, or a carboxyl group that can react with an inorganic or organic base to form a pharmaceutically acceptable salt. Pharmaceutically acceptable acid addition salts are formed from pharmaceutically acceptable acids, as is well known in the art. Examples of such salts include the pharmaceutically acceptable salts listed in Journal of Pharmaceutical Science, 66, 2-19 (1977) and The Handbook of Pharmaceutical Salts; Properties, Selection, and Use. PHStahl and CGWermuth (eds.), Verlag, Zurich (Switzerland) 2002, which are incorporated herein by reference in their entirety.

[0080] Pharmaceutically acceptable salts, in non-limiting examples, include sulfates, citrates, acetates, oxalates, chlorides, bromides, iodides, nitrates, hydrogen sulfates, phosphates, acidic phosphates, isonicotinates, lactates, salicylates, acidic citrates, tartrates, oleates, tannates, pantothenates, hydrogen tartrates, ascorbic acid, succinates, maleates, gentisinates, fumarates, glucons, glucarones, sugars, formates, benzoates, glutamates, methanesulfonates, ethanesulfonates, benzenesulfonates, p-toluenesulfonates, camphorsulfonates, pamoates, phenylacetates, trifluoroacetates, acrylates, chlorobenzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates, methylbenzoates, and o-acetates. Examples include xybenzoate, naphthalene-2-benzoate, isobutyrate, phenylbutyrate, α-hydroxybutyrate, butyn-1,4-dicarboxylate, hexyn-1,4-dicarboxylate, caprirate, caprylate, cinnamate, glycolate, heptanoate, hippurate, malate, hydroxymaleate, malonate, mandelate, mesylate, nicotinate, phthalate, teraphthalate, propiorate, propionate, phenylpropionate, sebacinate, suberinate, p-bromobenzenesulfonate, chlorobenzenesulfonate, ethylsulfonate, 2-hydroxyethylsulfonate, methylsulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, naphthalene-1,5-disulfonate, xylenesulfonate, and tartarate salt.

[0081] The term "pharmaceutically acceptable salt" also refers to salts of the composition of the present invention having an acidic functional group such as a carboxylic acid functional group and a base. Suitable bases include, but are not limited to, alkali metal hydroxides such as sodium, potassium, and lithium; alkaline earth metal hydroxides such as calcium and magnesium; hydroxides of other metals such as aluminum and zinc; ammonia, as well as unsubstituted or hydroxysubstituted mono, di, or trialkylamines, dicyclohexylamines; tributylamine; pyridine; N-methyl-N-ethylamine; diethylamine; triethylamine; mono-, bis-, or tris-(2-OH-lower alkylamines), such as mono-; bis-, or tris-(2-hydroxyethyl)amine, 2-hydroxy-tert-butylamine, or tris-(hydroxymethyl)methylamine; N,N-di-lower alkyl-N-(hydroxyl-lower alkyl)amine, such as N,N-dimethyl-N-(2-hydroxyethyl)amine or tri-(2-hydroxyethyl)amine; N-methyl-D-glucamine; and amino acids such as arginine and lysine.

[0082] In some embodiments, the compositions described herein are in the form of pharmaceutically acceptable salts.

[0083] How to use In one embodiment, a method is provided for the treatment of a patient or subject who is or has been receiving cancer treatment, or who has received or has received a bone marrow transplant, and / or who has been acutely exposed to a bone marrow suppressive dose of radiation, comprising administering a therapeutically effective amount of the recombinant human GM-CSF protein or a pharmaceutical composition thereof to the patient. In some embodiments, the patient is treated by modulating the clonal proliferation, survival, differentiation, and activation state of hematopoietic progenitor cells. In some embodiments, the patient is treated by modulating the bone marrow monocyte lineage by promoting the proliferation of megakaryocyte progenitor cells and erythrocyte progenitor cells. In some embodiments, the patient is treated by modulating hematopoietic progenitor cells by stimulating the survival, proliferation, and activation of neutrophils, macrophages, and / or dendritic cells. In some embodiments, the patient receives treatment by modulating hematopoietic progenitor cells by stimulating the survival, proliferation, and activation of neutrophils, macrophages, and / or dendritic cells after a bone marrow transplant.

[0084] In one embodiment, a therapeutic method is provided, comprising administering to a patient a therapeutically effective amount of the recombinant human GM-CSF protein or a pharmaceutical composition thereof, or contacting cells with a therapeutically effective amount of the pharmaceutical composition described herein and administering the cells thereof, wherein the therapeutic method promotes neutrophil recovery and reduces the incidence of infection after the introduction of chemotherapy; aggregates hematopoietic progenitor cells in the peripheral blood by leukocyte apheresis and transplantation; promotes bone marrow rearrangement after autologous or allogeneic bone marrow or peripheral blood progenitor cell transplantation; treats delayed neutrophil recovery or graft failure after autologous or allogeneic bone marrow transplantation; and / or treats hematopoietic syndrome of acute radiation syndrome (H-ARS).

[0085] In one embodiment, a method for treating a viral infection is provided, comprising administering to a patient in need of such treatment a composition comprising the recombinant human GM-CSF protein, or a composition comprising a pharmaceutical composition comprising the protein.

[0086] In some embodiments, the viral infection is influenza infection (optionally selected from influenza A, B, C, and D virus infections).

[0087] In some embodiments, the viral infection is a coronavirus infection. In some embodiments, the coronavirus is a beta-coronavirus (optionally selected from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), SARS-CoV, Middle East respiratory syndrome coronavirus (MERS-CoV), HCoV-HKU1, and HCoV-OC43). In some embodiments, the coronavirus is an alpha-coronavirus (optionally selected from HCoV-NL63 and HCoV-229E).

[0088] The above coronaviruses are members of the Coronaviridae family, which includes beta-coronaviruses and alpha-coronavirus respiratory pathogens that have only recently become known to invade humans. The Coronaviridae family includes severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), SARS-CoV, Middle East respiratory syndrome coronavirus (MERS-CoV), HCoV-HKU1, and HCoV-OC43. Alpha-coronaviruses include, for example, HCoV-NL63 and HCoV-229E.

[0089] Coronaviruses enter host cells via a "spike" surface glycoprotein that facilitates viral recognition by angiotensin-converting enzyme 2 (ACE2), a transmembrane receptor on mammalian hosts. (Zhou et al., A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature 2020.) This is the cause of COVID-19.

[0090] SARS-CoV-2 is a novel virus thought to originate from bats. COVID-19 causes severe respiratory distress, and this RNA virus strain is responsible for the recent outbreak, which has been declared a major threat to public health and a global emergency. Phylogenetic analysis of the whole genome of SARS-CoV-2 has revealed that this virus is most closely related to the SARS-like coronavirus group (Betacoronavirus genus, Sarbecovirus subgenus) (nucleotide similarity 89.1%). Wu et al., A new coronavirus associated with human respiratory disease in China. Nature, Feb 3, 2020.

[0091] SARS-CoV-2, also known as the S protein, is an enveloped single-stranded RNA virus that encodes a "spike" protein, a surface glycoprotein that mediates binding to cell surface receptors; an intrinsic membrane protein; an envelope protein; and a nucleocapsid protein. The S protein, including the S1 and S2 subunits, is a trimer class I fusion protein present in the prefusion conformation, undergoing structural changes to fuse the viral membrane with the host cell membrane. For example, see Li, F. Structure, Function, and Evolution of Coronavirus Spike Proteins. Annu. Rev. Virol. 3:237-261 (2016), the entire document of which is incorporated herein by reference. The structure of the SARS-CoV-2 spike protein in the prefusion conformation has been found. See Daniel et al., Cryo-EM structure of the SARS-CoV-2 spike in the prefusion conformation. Science, 19 Feb 2020, the entire document of which is incorporated herein by reference.

[0092] Phylogenetic analysis of the entire genome of SARS-CoV-2 (GenBank accession number: MN908947) revealed that this virus is most closely related to the SARS-like coronavirus group (Betacoronavirus genus, Sarbecovirus subgenus) (nucleotide similarity 89.1%). Wu et al., A new coronavirus associated with human respiratory disease in China. Nature, Feb 3, 2020 (the entire article is incorporated herein by reference).

[0093] The above-mentioned SARS-CoV-2 possesses spike surface glycoprotein, membrane glycoprotein M, envelope protein E, and nucleocapsidrin protein N. The entire genome (29,903 nucleotides, single-stranded RNA) of the above-mentioned SARS-CoV-2 coronavirus is listed in the NCBI database as GenBank reference sequence: MN908947. The above-mentioned coronavirus proteins can be selected from the group consisting of coronavirus spike protein (GenBank reference sequence: QHD43416), coronavirus membrane glycoprotein M (GenBank reference sequence: QHD43419), coronavirus envelope protein E (GenBank reference sequence: QHD43418), and coronavirus nucleocapsidrin protein E (GenBank reference sequence: QHD43423).

[0094] In some embodiments, this method prevents or mitigates the onset of acute respiratory distress syndrome (ARDS) in patients.

[0095] In some embodiments, the coronavirus is SARS-CoV-2. In some embodiments, the patient is suffering from COVID-19. In some embodiments, the patient is suffering from one or more of the following: fever, cough, shortness of breath, diarrhea, upper respiratory tract symptoms, lower respiratory tract symptoms, pneumonia, and acute respiratory syndrome.

[0096] In some embodiments, the patient is hypoxic. In some embodiments, the patient is experiencing dyspnea. In some embodiments, the method improves oxygenation in the patient. In some embodiments, the method prevents or mitigates the transition from respiratory distress to cytokine imbalance in the patient. In some embodiments, the method reverses or prevents a cytokine storm. In some embodiments, the method reverses or prevents a cytokine storm in the lung or systemically. In some embodiments, the cytokine storm is selected from one or more of systemic inflammatory response syndromes, cytokine release syndromes, macrophage activation syndromes, and hemophagocytic lymphohistiocytosis. In some embodiments, the method reverses or prevents the excessive production of one or more inflammatory cytokines. In some embodiments, the inflammatory cytokine is one or more of IL-6, IL-1, IL-1 receptor antagonist (IL-1ra), IL-2ra, IL-10, IL-18, TNFα, interferon-γ, CXCL10, and CCL7.

[0097] In some embodiments, this method reduces the viral load in the patient compared to before treatment.

[0098] In one embodiment, a method for treating or preventing a viral infection in a subject requiring the method is provided, comprising: preparing plasma derived from a donor subject who has recovered from the viral infection (provided that the plasma contains IgG, IgM, and / or IgA antibodies against the virus causing the infection, and the donor subject has been treated with recombinant human GM-CSF as described herein to stimulate the production of the antibodies); and administering the plasma to the subject requiring the method. In one embodiment, a method for treating or preventing a viral infection in a subject requiring the method is provided, comprising: administering recombinant human GM-CSF as described herein to a donor subject who has recovered from the viral infection; isolating plasma containing IgG, IgM, and / or IgA antibodies against the virus causing the infection from the donor subject; and administering the plasma to the subject requiring the method.

[0099] In some embodiments, such a method provides passive immunity to the virus to a subject requiring the method.

[0100] In some embodiments, the IgG, IgM, and / or IgA antibodies specifically bind to viral antigens. In some embodiments, the IgG, IgM, and / or IgA antibodies neutralize the virus. In some embodiments, the IgG, IgM, and / or IgA antibodies prevent or reduce infection of cells by the virus.

[0101] In some embodiments, the viral infection is a beta-coronavirus infection, optionally selected from the beta-coronavirus infections selected from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, severe acute respiratory syndrome coronavirus (SARS-CoV-1) infection, Middle East respiratory syndrome coronavirus (MERS-CoV) infection, HCoV-HKU1 infection, and HCoV-OC43 infection. In some embodiments, the viral infection is an alpha-coronavirus infection, optionally selected from the alpha-coronavirus infections selected from HCoV-NL63 infection and HCoV-229E infection.

[0102] In some embodiments, the above-mentioned beta-coronavirus infection is severe acute respiratory syndrome (SARS).

[0103] In some embodiments, the above-mentioned beta-coronavirus infection is or is related to coronavirus disease 2019 (COVID-19).

[0104] In some embodiments, the viral infection is an influenza virus infection, which is optionally selected from influenza A, B, C, and D type virus infections. In some embodiments, the influenza infection is the 2009 pandemic influenza A (H1N1) or avian influenza A (H5N1).

[0105] In some embodiments, the donor subject tested positive for the viral infection prior to recovery. In some embodiments, the symptoms of the viral infection had disappeared in the donor subject prior to donation. In some embodiments, the donor subject tested positive for antibodies against the virus using serological testing. In some embodiments, the donor subject exhibits a measurable neutralizing antibody titer. In some embodiments, the neutralizing antibody titer is at least about 1:160. In some embodiments, the plasma is isolated from a blood sample derived from the donor subject. In some embodiments, the plasma is isolated by plasma exchange. In some embodiments, the plasma contains a therapeutically effective amount of IgG, IgM, and / or IgA antibodies against the virus causing the infection.

[0106] Combination therapy and additional treatments In various embodiments, the pharmaceutical composition of the present invention is administered concurrently in combination with further agents(s). Concurrent administration may be simultaneous or sequential.

[0107] In one embodiment, the further therapeutic agent of the present invention and GM-CSF are administered simultaneously to a subject. As used herein, “simultaneously” means that the further therapeutic agent and GM-CSF are administered at time intervals not exceeding about 60 minutes, for example, not exceeding about 30 minutes, not exceeding about 20 minutes, not exceeding about 10 minutes, not exceeding about 5 minutes, or not exceeding about 1 minute. The administration of the further therapeutic agent and GM-CSF may be by simultaneous administration of a single formulation (e.g., a formulation comprising the further therapeutic agent and the GM-CSF composition) or by simultaneous administration of separate formulations (e.g., a first formulation comprising the further therapeutic agent and a second formulation comprising the GM-CSF composition).

[0108] Co-administration is not necessary if the timing of administration of the therapeutic agents overlaps in time with the pharmacological activity of the additional therapeutic agent and GM-CSF, thereby producing a combined therapeutic effect. For example, the additional therapeutic agent and the targeted portion, GM-CSF composition may be administered sequentially. As used herein, "sequentially" means that the additional therapeutic agent and GM-CSF are administered at time intervals of more than approximately 60 minutes. For example, the time between sequential administration of the additional therapeutic agent and GM-CSF may be more than approximately 60 minutes, more than approximately 2 hours, more than approximately 5 hours, more than approximately 10 hours, more than approximately 1 day, more than approximately 2 days, more than approximately 3 days, more than approximately 1 week, more than approximately 2 weeks, or more than approximately 1 month. The optimal administration time will depend on the rate of metabolism, excretion, and / or the pharmacodynamic activity of the additional therapeutic agent and GM-CSF being administered. Either the additional therapeutic agent or the GM-CSF composition may be administered first.

[0109] Simultaneous administration does not require that the above-mentioned therapeutic agents be administered to the target via the same route of administration. Conversely, each therapeutic agent may be administered via any and appropriate route, such as parenteral or oral administration.

[0110] In some embodiments, the GM-CSF described herein acts synergistically when administered concurrently with another therapeutic agent. In such embodiments, the targeted portion, the GM-CSF composition, and the further therapeutic agent may be administered at lower doses than those used when the agent is used as monotherapy.

[0111] In some embodiments, the further therapeutic agent is an antiviral drug.

[0112] In some embodiments, the above further therapeutic agents include remdesivir, favipiravir, oseltamivir, baloxavir, galidesivir, amprenavir, tipranavir, saquinavir, nelfinavir, indinavir, darunavir, atazanavir, emetine, lopinavir, and / or ritonavir, arbidol, and lopinavir / ritonavir, and / or ribavirin, darunavir, and cobicistat, and / or IFN-beta-1b, β-D-N4-hydroxycytidine (NHC), e.g., EIDD-1931 or EIDD-2801 or EIDD-2801; glucocorticoids, IFN-a 2a, IFN-a Immunomodulators such as 2b, IFN-b, pegylated IFN-g, baricitinib, sirolimus, crazakizumab, canakinumab, XPro1595, tocilizumab, sarilumab, siltuximab, adalimumab, eculizumab, ivermectin, anakinra, prezcobix, xiyanping, fingolimod, methylprednisolone, leronlimab, thalidomide, MK-2206, nicolasamide, nitazoxamide, chloroquine, or hydroxychloroquine; Kalimycin, brilacidine, azithromycin The drugs are selected from antibiotics such as romycin and valinomycin; angiotensin inhibitors / antagonists such as rhACE2 / GSK2586881 / APN01, losartan, eprosartan, telmisartan, and valsartan; serine protease inhibitors including camostat mesylate and nafamostat; and other drugs such as bromhexine, aprotinin, chlorpromazine, zotatifin, methotrexate, lenalidomide, anti-VEGF-A, and intravenous immunoglobulin (IVIG). For example, in some embodiments, any of these additional therapeutic agents are used in relation to SARS-CoV-2 infection.

[0113] In some embodiments, the above further therapeutic agents are selected from favipiravir, laninamivir octanoate, peramivir, zanamivir, oseltamivir phosphate, baloxavir marboxil, umifenovir, urumin, amantadine hydrochloride, rimantadine hydrochloride, adapromin, LASAG / BAY81-87981, celecoxib, etanercept, metformin, gemcitabine, dapivine, trametinib, lisinopril, naproxen, nalidixic acid, dorzolamide, ruxolitinib, midodrine, diltiazem; statins including atorvastatin and nitazoxanide; and PPAR antagonists including gemfibrozil. For example, in some embodiments, any of these further therapeutic agents are used in relation to influenza infection.

[0114] array Sequence ID 1 is wild-type GM-CSF.

[0115] [ka]

[0116] Sequence ID 2 is salglamostim.

[0117] APARSPSPSTQPWEHVNAIQEALRLLNLSRDTAAEMNETVEVISEMFDLQEPTCLQTRLELYKQGLRGSLTKLKGPLTMMASHYKQHCPPTPETSCATQIITFESFKENLKDFLLVIPFDCWEPVQE.

[0118] definition The following definitions are used in connection with the inventions disclosed herein. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the inventions pertain.

[0119] "Effective dose" is the amount that is effective in treating or alleviating coronavirus infection when used in conjunction with drugs that are effective in treating coronavirus infection.

[0120] "A," "an," or "the," as used herein, can mean one or more. Furthermore, the term "about," when used in relation to a numerical value being mentioned, means the numerical value being mentioned ± up to 10% of the numerical value being mentioned. For example, the phrase "about 50" covers a range of 45 to 55.

[0121] All percentages of composition, where referred to herein, are based on the weight of the total composition unless otherwise specified. The words “including” and their variations, as used herein, are intended to be non-limiting, and not to exclude other similar items that may be similarly useful in the materials, compositions, devices, and methods of this art. Similarly, the terms “can” and “may” and their variations are non-limiting, and any statement that an embodiment may include or be including certain elements or features is not intended to exclude other embodiments of the art that do not include those elements or features.

[0122] The open-ended term "comprising," as a synonym for terms such as including, containing, or having, is used herein to describe and claim the invention, the present invention, or embodiments thereof, but alternative terms such as "consisting of" or "consisting essentially of" may be used.

[0123] The present invention can be further described by the following non-limiting embodiments. [Examples]

[0124] Example 1: List of additional components during fermentation Production fermentation was carried out incorporating the addition of Bacto-Pepton, a complex material, and key components contained in yeast extract. The list of additional components during fermentation included MgSO4, KH2PO4, CaCl2, adenine, MEM vitamin solution, and YNB trace element solution. Biomass and productivity increased significantly in fermentation carried out in the presence of all additional components and trace element solutions alone, indicating that the trace element solution contains key components for increasing the productivity and culture biomass of recombinant human (rhu) GM-CSF. The above trace element solution contains six elements: copper, molybdate, zinc, iron, boric acid, and manganese. To identify which of these elements is responsible for the increase in productivity and biomass, the six trace elements were individually screened during production fermentation at concentrations matching those in the YNB trace element solution (the final concentration in the fermenter for each screened element), as shown in Table 1. [Table 1]

[0125] Example 2: Biochemical assay of trace element addition Dissolved Oxygen Profile: The dissolved oxygen level was continuously monitored as a process parameter during production fermentation. This level served as a surrogate for the oxygen uptake of the yeast culture and indicated yeast culture growth. The dissolved oxygen profiles of production fermentation carried out in the presence of each trace element are shown (Figure 1A). The oxygen uptake of the yeast culture was significantly higher in the batch to which copper (copper sulfate / CuSO4) was added, resulting in a decrease in dissolved oxygen levels. A dissolved oxygen cascade control method was used to prevent the dissolved oxygen level from falling below the suppression level.

[0126] In Figure 1B, the dissolved oxygen profile of production fermentation with copper supplementation is compared to the same profile of a scaled-down commercial process (without supplementation). This result shows a significant difference in oxygen requirements during yeast culture with copper supplementation.

[0127] Cell wet weight profile: Yeast culture biomass was evaluated as wet cell weight (WCW). WCW was measured by centrifugation of 20 mL of cell culture medium in a pre-weighed 50 mL centrifuge tube. The supernatant was aspirated and removed, and the centrifuge tube was re-weighed to calculate the WCW of each production fermentation batch. The WCW of production fermentation carried out in the presence of each trace element is shown (Figure 2A), and the highest biomass value was obtained with copper addition. When comparing copper addition with a scaled-down commercial process (without addition), the biomass was significantly higher in the copper-added fermentation than in the scaled-down commercial fermentation (Figure 2B).

[0128] Example 3: Comparison of Recombinant Human GM-CSF Potency and Glycoform The concentration of recombinant human (rhu) GM-CSF in test samples was measured using reversed-phase HPLC with a C18 column and a gradient of acetonitrile maintaining a constant composition of 0.2 M sodium chloride throughout the entire gradient program. Trifluoroacetic acid (TFA) was used as an ion pairing agent (0.1 vol%) in each mobile phase solvent. The results for rhu GM-CSF concentrations in the test samples were interpolated using a 6-step external standard calibration curve prepared from a GM-CSF reference standard. Figure 3 shows a significant increase in rhu GM-CSF concentration compared to a scaled-down commercial process (without additives).

[0129] The reverse-phase HPLC procedure used to measure the rhu GM-CSF concentration separated the rhu GM-CSF glycosylation variants into three major glycoform groups via a C18 column. The four target peaks were integrated and quantified, and their respective compositions are described below. Peak 1 = GM-CSF related impurities (oxidation). (Note: In the sample before C4 purification, there is an excessively glycosylated peak that obscures the true peak 1.) Peak 2 = N- and (N+O) bonded glycoforms Peak 3 = O-linked glycoform Peak 4 = Non-glycosylated GM-CSF Table 2 shows that the glycosylation variants derived from fermentation with copper addition (ratio of peaks 2-4) (an indicator of product quality) are comparable to the historical average values ​​of the commercial process. The ratio of glycosylation variants obtained with copper addition is within a 95% tolerance, covering 99.73% of the entire production history of commercial rhu GM-CSF, indicating that copper addition does not affect the quality characteristics of GM-CSF glycoforms or the product.

[0130] Table 2 shows the glycoform profiles (expressed as peak ratios) of recombinant human GM-CSF obtained by an exogenous copper-added fermentation process, compared to a scaled-down version of a historical commercial process. This table compares the peak ratios of copper-added fermenters with the average values ​​and commercial process criteria of the historical commercial process. [Table 2]

[0131] Example 4: Comparison of results from in-process release tests and steady-state release tests for trace element addition. Data analysis was performed on C4 purification (Table 3), C18 purification (Table 4), and bulk drug substance (BDS). The above data demonstrate that production fermentation with added copper produces a substance comparable to that produced by current commercial manufacturing processes.

[0132] A key indicator of the product quality of the protein after downstream processing is the glycoform ratio, measured by the T-0075 assay. Peaks 2, 3, and 4 represent glycosylated variants of salglamostim, while peak 1 represents an over-glycosylated impurity. Peak 1 is removed by the C4 purification unit operation. The results of the CuSO4-added BDS process validation (CuSO4PV) of the in-process and BDS glycoforms are equivalent to those of commercial in-process and BDS lots (BDS 6-8).

[0133] Table 3 provides an overview of the equivalence of glycoform ratios. Table 3 outlines the equivalence of C4 purified glycoform ratios in the copper-added fermentation process compared to historical commercial processes. The batch numbers listed in Tables 3 and 4 correspond to the C4 purified PV operations. [Table 3]

[0134] Table 4 summarizes the equivalence of the C18 purified glycoform ratio of the copper-added fermentation process compared to historical commercial processes. [Table 4]

[0135] Table 5 summarizes the equivalence of BDS glycoform ratios in copper-added fermentation processes compared to scaled-down historical commercial processes. [Table 5]

[0136] Example 5: Comparison of BDS release tests with trace element additions The results of BDS release tests for three commercial BDSs and one process validation BDS all met the current specifications for BDS release. All results confirm that the salglamostim protein (CuSO4PV) generated during the validation run of the copper-added process is equivalent to the results from chronological commercial runs (BDS 6-8).

[0137] Figure 4 shows the results of the SDS-PAGE-silver staining (T-0002) assay used to evaluate impurities in salglamostim BDS resulting from protein degradation or contamination by substances other than protein products. The impurity test results for CuSO4 batch (CuSO4PV) in the BDS are comparable to those of commercial BDS batches 6-8.

[0138] Figure 5 shows the results of a concentration measurement test (T-0013) conducted to evaluate the protein purity level of Sarglamostim BDS. The protein purity test results for the CuSO4 batch (CuSO4PV) in BDS are equivalent to those of commercial BDS batches 6-8.

[0139] Figure 6 shows the isoelectric focusing results (T-0114) used to identify the identity of Salglamostim BDS. The isoelectric focusing test results for the CuSO4 batch (CuSO4PV) in the BDS are comparable to those of commercial BDS batches 6-8.

[0140] Tables 6 and 7 further summarize the results of the BDS release test. [Table 6] [Table 7]

[0141] Example 7: Characterization of product proteins produced by a CuSO4-added manufacturing process compared to an authorized commercial process. Further product and process characterization was performed to confirm that the product protein produced in the manufacturing process with CuSO4 addition is equivalent to that produced in the approved commercial process. For product characterization, assays were performed to evaluate the protein's composition and structure in detail. The results confirm that the salglamostim protein (CuSO4PV batch, CuSO4PV) produced during process validation runs is equivalent to the results of commercial BDS runs BDS 6-8. Table 8 summarizes the product characterization results. [Table 8-1] [Table 8-2]

[0142] Process characterization consists of evaluating the removal of residual process components (RPCs) throughout the entire downstream operation. Analysis of CuSO4PV batch (CuSO4PV) samples revealed that RPCs were removed throughout the purification process. The RPC levels of the CuSO4 batch in the BDS were comparable to those of recent batches and all chronological batches. The results support that the RPC removal level of the CuSO4 addition process was comparable to that of the current manufacturing process. The results are shown in Figure 7 and Table 9. [Table 9]

[0143] Trypsin peptide map (TCPK-trypsin) 1C-terminal analysis was performed using the following method: rhu GM-CSF was enzymatically digested with trypsin and reduced. The resulting peptides were separated by RP-HPLC. Three major C-terminal peptides were analyzed by retention time and quantified by normalized area ratio. The results of the C-terminal analysis showed that the CuSO4 batch (CuSO4PV) in BDS was equivalent to the commercial BDS batch (BDS 6-8). The results are shown in Figure 8 and Table 10. [Table 10]

[0144] Disulfide crosslinking is measured by peptide mapping with Glu-C at low pH. The low pH is necessary to prevent disulfide reconstitution. Two major peaks, 11 and 12, contain the expected disulfide crosslinked peptides (G7-8=G10 and G9=G11-13 / G9=G12-13, respectively). Peptide fragments were confirmed by mass spectrometry. The results of the disulfide crosslinking analysis demonstrate equivalence between the CuSO4 batch (CuSO4PV) and commercial BDS batches (BDS 6-8) in the BDS batch. The results are shown in Table 11 and Figure 9. [Table 11]

[0145] The size of the chain bound to nitrogen at the N27 site (site occupancy at N27) was measured by peptide mapping using Glu-C at low pH, after removing both oligosaccharides bound to nitrogen and oxygen (using PNGase and alpha-mannosidase, respectively). In the removal of oligosaccharides bound to nitrogen, the enzyme PNGase converts the asparaginyl residue bound to nitrogen to an aspartyl residue, and the resulting deamidated fragment can be quantified by RP-HPLC. Using this method, the following formula was obtained:

number

[0146] The total O-glycosylated glycoforms were quantified by comparing peptide maps obtained using Glu-C without the use of alpha-mannosidase. The size (site occupancy) of the total O-linked glycosylated chains was expressed by the following formula:

number

[0147] The Glu-C peptide map fragment G9 (residues 61-93) consists of two methionines (M 79 and M 80) is included. Methionine 79, which is oxidized, elutes before the G9 peak and is thus detectable on the RP-HPLC chromatogram (previously measured by ESI-MS / MS). Methionine 80 is not observed, but it cannot be completely excluded. The ratio of oxidation at methionine 79 indicates that the CuSO4 batch (CuSO4PV) in BDS is equivalent to the results in commercial BDS batches 6 - 8. The results are shown in Table 14 and Figure 12.

Table 14

[0148] The tertiary structure of the above protein was measured by using internal fluorescence and measuring the shift of the emission maximum wavelength as a function of temperature, and the thermal stability of the lot was monitored. Fluorescence spectra and thermal unfolding data (T m ) show the equivalence among the four BDS lots (CuSO4PV and commercial BDS batches (BDS 6 - 8)) tested. The results are shown in Figures 13 and 14 and Table 15.

Table 15

[0149] Circular dichroism (CD) spectroscopy was used to measure the secondary structure, melting temperature (T m ), and the onset of protein unfolding (T 開始 ) based on the absorption difference between left and right circularly polarized light as a function of temperature. The minimum absorbance at 208 nm and 222 nm in the CD scan indicates that the structure is mainly an alpha helix among the four BDS lots. CD scan and thermal denaturation data (T m and T 開始 ) show that the CuSO4 batch (CuSO4PV) in BDS is equivalent to the results in commercial BDS batches 6 - 8. The results are shown in Figure 15, and Tables 16, 17, and 18.

Table 16

[0150] MALDI-MS (matrix-assisted laser desorption / ionization mass spectrometry) intact mass spectrometry is a method that can provide data on structural integrity and protein modification by comparing the observed spectral mass with the theoretical molecular weight based on the amino acid sequence (SEQ ID NO: 2) and associated modifications of salglamostim.

[0151] MALDI-MS was performed using an Applied Biosystems 4800 MALDI-TOF / TOF. Samples were diluted 10-fold with sinapic acid, spotted onto MALDI plates, and MS was acquired for 15 minutes per sample ranging from 2 to 19 kDa.

[0152] Intact MALDI-MS was used to determine the molecular weights of salglamostim and glycan across lots. Figure 16 shows the complete MALDI mass spectra for 12–19 kDa, Figure 17 shows salglamostim for 14–16 kDa, and Figure 18 shows salglamostim + glycan for 16–19 kDa. The corresponding assignments of the observed mass peaks are shown in Table 19.

[0153] These results show equivalent MALDI-MS profiles and masses, confirming the equivalence of these proteins and glycans between the results from the CuSO4 batch (CuSO4PV) in BDS and the results from commercial BDS batches 6–8. [Table 19-1] [Table 19-2]

[0154] Proteomics LC-MS / MS analysis of host cell proteins (HCPs) is a method for comprehensively identifying and quantifying low-abundance proteins in a sample. To identify HCPs in commercial lots BDS 6-8 and CuSO4-added PV lots (PVs), the above BDSs were proteolytically cleaved with trypsin and separated by reverse-phase C18 nano-LC with a 60-minute gradient. Tandem mass spectra of the LC peaks were obtained using an Orbitrap Elite ETD mass spectrometer, and protein identity was detected using the Protein Metrics database and spectral analysis software. The relative amounts of yeast HCPs in each sample were obtained from the extracted ion signals (XIC) of each peptide and compared between lots for this analysis.

[0155] Table 20 lists proteins identified with an XIC area of ​​0.01% or more. For the XIC area ratio, the upper number in each cell represents the relative value to all identified proteins. The lower number in parentheses indicates the relative value after method-induced artifacts and contaminants have been removed.

[0156] The results above indicate that all lots contain at least 99% salglamostim by ion signaling, which suggests that most HCPs are removed during the purification step. Furthermore, the identified low-abundance HCPs are comparable across lots. Therefore, the HCP profiles identified in CuSO4-added PV lots (CuSO4PV) in BDS are comparable to the results from commercial BDS batches 6-8. [Table 20-1] [Table 20-2]

[0157] Testing for elemental impurities (including copper) was performed on two commercial BDS batches (BDS 7 and BDS 8) and a CuSO4-added process validation BDS batch (BDS PV) using quantitative screening elements by ICP-MS, selected for testing in accordance with the ICH Q3D(R1) recommendations, specifically the guidance on elemental impurities (March 22, 2019). Additionally, molybdenum, intentionally added in trace amounts to the process, was included in the test design. The results (Table 21) show that the impurity profile of the process validation batch is consistent with recent commercial batches, and these results are reported below the limit of quantification (LOQ) of the assay. Although Cu was introduced during the upstream cell culture process, the data showed that elemental impurities, including Cu, decreased (as expected) during subsequent downstream processing. All results were below the limits of the maximum permissible concentration (MPC) and control threshold (CT). All results correspond to below the permissible daily exposure levels for parenteral drugs as described in ICH Q3D. [Table 21]

[0158] Assuming an increase in biomass due to the CuSO4 process, a Threshold DNA test was conducted to verify that the residual DNA level in BDS met the requirements. The criteria were based on historical BDS specifications (Note: Residual DNA was removed from the product release criteria in accordance with the August 2020 management change MOC-00074). The results of the process-validated BDS CuSO4 batch in BDS(PV) met the criteria. See Table 22 below. [Table 22] *To eliminate slight positive bias in the mean amount of DNA-free samples, the standard curve for the Threshold DNA assay uses an extended power fit algorithm that forces the regression line to zero, which can result in the quantification of near-zero negative values.

[0159] Equivalents Those skilled in the art will be able to recognize numerous equivalents to the specific embodiments described herein, or to verify such equivalents by conventional experimentation alone. Such equivalents are intended to be covered by the appended claims.

[0160] support All patents and publications referenced herein are incorporated herein by reference in their entirety by this statement.

[0161] All headings used herein are for organizational purposes only and are not intended to limit the disclosure in any way. The content of any individual section may apply equally to all sections.

Claims

1. 1. A method for producing a recombinant protein, comprising: the recombinant protein is recombinant human granulocyte macrophage colony stimulating factor (rhu GM-CSF) protein; The manufacturing method includes: (a) supplementing a culture medium containing a host cell that comprises a nucleic acid molecule encoding said recombinant protein and that has the ability to produce said recombinant protein during fermentation with trace elements; (b) isolating the recombinant protein; and Including, The method of manufacturing, wherein the trace elements are exogenously added to the medium to supplement the amount of trace elements in the medium.

2. the recombinant human granulocyte macrophage colony stimulating factor (rhu GM-CSF) protein comprises an amino acid sequence having at least about 97% identity to SEQ ID NO:1 or SEQ ID NO:2; and / or 2. The method of claim 1, optionally wherein the recombinant protein binds to and / or activates the granulocyte-macrophage colony-stimulating factor receptor (GM-CSF-R-alpha or CSF2R).

3. the addition of said trace element during the production of said recombinant protein increases the expression level of said recombinant protein compared to a process without the addition of said trace element; or Addition of said trace element during the production of said recombinant protein improves the fermentation yield of said recombinant protein compared to a process without the addition of said trace element, or The addition of the trace elements improves the consistency of fermentation performance during the production of the recombinant protein compared to methods without the addition of the trace elements. The method of claim 1.

4. The trace element is copper, Optionally, the copper is in the form of a copper derivative or a copper compound; Optionally, the copper is a copper salt; Optionally, the copper salt is cupric sulfate or copper sulfate; and / or 2. The method of claim 1, optionally wherein copper is added to the medium in an amount of about 0.5 μM to about 100 μM (optionally about 0.5 μM to about 80 μM, or optionally about 1 μM to about 20 μM).

5. The method according to any one of claims 1 to 4, wherein the nucleic acid molecule is a vector or has a codon-optimized sequence.

6. the host cell expresses the recombinant protein; Optionally, the host cell is a non-human host cell; Optionally, the non-human host cell is a yeast cell or a mammalian cell (optionally a Chinese Hamster Ovary (CHO) cell); Optionally, the yeast cell is a non-methylotrophic yeast cell; Optionally, the host cell is a Saccharomyces cerevisiae cell. The method according to any one of claims 1 to 4.

7. A pharmaceutical composition comprising recombinant human GM-CSF obtained using the method according to any one of claims 1 to 4, and a pharma- ceutically acceptable excipient or carrier.

8. 8. The pharmaceutical composition of claim 7 for treating a patient or subject who is or has been treated for cancer, or who is or has been treated for an infectious agent and / or has been treated to treat the effects of an infectious disease, or who is or has been treated for a bone marrow transplant, and / or has been acutely exposed to a myelosuppressive dose of radiation.

9. The patient is treated by modulating the clonal expansion, survival, differentiation, and activation state of hematopoietic progenitor cells; or Optionally, the patient is treated by modulating the myelomonocytic lineage by promoting proliferation of megakaryocyte and erythroid progenitor cells; or Optionally, the patient is treated by modulating hematopoietic progenitor cells by stimulating survival, proliferation, and activation of neutrophils, macrophages, and / or dendritic cells; or 9. The pharmaceutical composition of claim 8, optionally wherein the patient is treated by regulating hematopoietic progenitor cells by stimulating survival, proliferation and activation of neutrophils, macrophages and / or dendritic cells after bone marrow transplantation.

10. Administering to a patient a therapeutically effective amount of the pharmaceutical composition of claim 7; or contacting a cell with an effective amount of said pharmaceutical composition and administering a therapeutically effective amount of said cell.

8. A pharmaceutical composition according to claim 7 for use in a method of treatment comprising: The treatment, Promoting neutrophil recovery and reducing the incidence of infections after the induction of chemotherapy; Concentrating hematopoietic progenitor cells in peripheral blood by leukapheresis and transplantation; Promoting bone marrow reconstitution following autologous or allogeneic bone marrow or peripheral blood progenitor cell transplantation; To treat delayed neutrophil recovery or graft failure following autologous or allogeneic bone marrow transplantation; Treating hematopoietic syndrome of acute radiation syndrome (H-ARS); and / or Treating the aftereffects and long-term effects of infection; Pharmaceutical compositions.

11. 8. The pharmaceutical composition of claim 7 for treating a viral infection, said treatment comprising administering to a patient in need thereof an effective amount of a composition comprising said pharmaceutical composition.

12. the virus is an influenza virus or a coronavirus, wherein the coronavirus is optionally a betacoronavirus, optionally selected from Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2), SARS-CoV, Middle East Respiratory Syndrome coronavirus (MERS-CoV), HCoV-HKU1, and HCoV-OC43, or an alphacoronavirus, optionally selected from HCoV-NL63 and HCoV-229E, optionally wherein the coronavirus is SARS-CoV-2, and optionally wherein the patient is suffering from COVID-19; and / or Optionally, the patient is suffering from one or more of fever, cough, shortness of breath, diarrhea, upper respiratory tract symptoms, lower respiratory tract symptoms, pneumonia, and acute respiratory syndrome; and / or Optionally, the patient is hypoxic; and / or Optionally, the patient is experiencing respiratory distress, and / or Optionally, said treating prevents or alleviates the onset of Acute Respiratory Distress Syndrome (ARDS) in said patient; and / or Optionally, the treatment improves oxygenation of the patient; and / or Optionally, said treating prevents or alleviates the transition from respiratory distress to cytokine imbalance in said patient; and / or Optionally, said treatment reverses or prevents a cytokine storm, optionally, said treatment reverses or prevents a cytokine storm in the lungs or systemically, and / or optionally, said cytokine storm is selected from one or more of systemic inflammatory response syndrome, cytokine release syndrome, macrophage activation syndrome, and hemophagocytic lymphohistiocytosis, and / or Optionally, the treatment reverses or prevents excessive production of one or more inflammatory cytokines, and optionally, the inflammatory cytokines are one or more of IL-6, IL-1, IL-1 receptor antagonist (IL-1ra), IL-2ra, IL-10, IL-18, TNFα, interferon-γ, CXCL10, and CCL7; and / or Optionally, the treatment reduces viral load in the patient compared to before treatment. The pharmaceutical composition according to claim 11.

13. The pharmaceutical composition of claim 7 for use in treating a viral infection in a subject in need thereof, said treatment comprising: Providing plasma from a donor subject who has recovered from the viral infection, the plasma contains IgG, IgM, and / or IgA antibodies against the virus causing the infection; and the donor subject is undergoing treatment with the pharmaceutical composition to stimulate production of the antibodies; and administering said plasma to said subject in need of said treatment.

13. A pharmaceutical composition comprising:

14. The pharmaceutical composition of claim 7 for use in treating a viral infection in a subject in need thereof, said treatment comprising: administering the pharmaceutical composition to a donor subject who has recovered from the viral infection; isolating plasma from the donor subject that contains IgG, IgM, and / or IgA antibodies against the virus causing the infectious disease; administering said plasma to said subject in need of said treatment; 13. A pharmaceutical composition comprising:

15. The method of claim 1, wherein the treatment provides a subject in need of the treatment with passive immunity against the virus; and / or Optionally, the IgG, IgM, and / or IgA antibodies specifically bind to a viral antigen; and / or Optionally, the IgG, IgM, and / or IgA antibodies neutralize the virus; and / or Optionally, the IgG, IgM, and / or IgA antibodies prevent or reduce infection of a cell by the virus; and / or Optionally, the viral infection is a betacoronavirus infection, optionally selected from a betacoronavirus infection selected from a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, a severe acute respiratory syndrome coronavirus (SARS-CoV-1) infection, a Middle East respiratory syndrome coronavirus (MERS-CoV) infection, an HCoV-HKU1 infection, and an HCoV-OC43 infection; and / or optionally, the viral infection is an alphacoronavirus infection, optionally selected from an alphacoronavirus infection selected from an HCoV-NL63 infection and an HCoV-229E infection; optionally, the betacoronavirus infection is severe acute respiratory syndrome (SARS); or optionally, the betacoronavirus infection is or is associated with coronavirus disease 2019 (COVID-19).

15. The pharmaceutical composition of claim 14.

16. the viral infection is an influenza infection, optionally selected from influenza A, B, C, and D virus infections, optionally wherein the influenza infection is 2009 pandemic influenza A (H1N1) or avian influenza A (H5N1); and / or Optionally, the donor subject tested positive for the viral infection prior to recovery; and / or Optionally, the donor subject is symptom-free of viral infection prior to donation; and / or Optionally, the donor subject has tested positive for antibodies to the virus using a serological test; and / or Optionally, the donor subject exhibits measurable neutralizing antibody titers, and / or Optionally, the neutralizing antibody titer is at least about 1:160; and / or Optionally, said plasma is isolated from a blood sample from said donor subject, optionally, said plasma is isolated by plasmapheresis; and / or Optionally, the plasma comprises a therapeutically effective amount of IgG, IgM, and / or IgA antibodies against the virus causing the infection.

15. The pharmaceutical composition of claim 14.

17. 1. A method for producing a recombinant protein, comprising: (a) adding a copper salt to a culture medium containing a host cell that contains a nucleic acid molecule encoding said recombinant protein and that has the ability to produce said recombinant protein during fermentation; (b) isolating the recombinant protein; and Including, the copper salt is exogenously added to the medium in an amount of about 1 μM to about 20 μM to supplement the amount of trace elements in the medium; the copper salt is cupric sulfate or copper sulfate, the recombinant protein is a recombinant human granulocyte macrophage colony stimulating factor (rhu GM-CSF) protein having at least about 97% identity to SEQ ID NO:2; Optionally, the addition of said trace element during the production of said recombinant protein increases the expression level of said recombinant protein compared to a method without the addition of said trace element.

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