Preparations containing G-CSF and their use

Abstract

Described herein are compositions of G-CSF dimers, including G-CSF-Fc dimers. Methods of treating or preventing diseases or conditions using the compositions, systems, kits, and preparation methods are also provided.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority from PCT / CN2022 / 113560, filed August 19, 2022, the entire contents of which are incorporated herein by reference.

[0002] Electronic Sequence Listing Reference The entire contents of the electronic sequence listing (720622001941SEQLIST.xml, size: 34,294 bytes, created on August 10, 2023) are incorporated herein by reference.

[0003] The present disclosure, in some aspects, relates to formulations (e.g., pharmaceutical compositions) of granulocyte colony-stimulating factor (G-CSF) and uses thereof. [Background technology]

[0004] The present embodiments relate generally to G-CSF formulations useful for injection and general administration.

[0005] G-CSF is a hematopoietic glycoprotein produced by stromal cells, macrophages, endothelial cells, fibroblasts, and monocytes. G-CSF binds to the G-CSF receptor (G-CSFR) expressed on precursor cells in the bone marrow to promote the proliferation and differentiation of various types of blood cells, such as neutrophils, which play an important role in the body's defense against bacterial infections. G-CSF can also activate mature neutrophils to participate in immune responses or act synergistically with other hematopoietic growth factors, such as stem cell factor, Flt-3 ligand, and GM-CSF, to exert hematopoietic functions.

[0006] G-CSF formulations can be administered to patients via subcutaneous injection. Both liquid and lyophilized dosage forms are available for pharmaceutical use. However, highly concentrated G-CSF protein formulations can present numerous challenges in formulation development, especially for liquid formulations. High protein concentrations can cause protein stability issues due to increased protein-protein interactions, leading to increased formation of soluble and insoluble protein aggregates. When the protein concentration approaches its apparent solubility limit, phase separation due to precipitation, gelation, and / or crystallization can occur. Furthermore, the high protein concentrations desired in formulations intended for intramuscular or subcutaneous administration and for convenience for healthcare providers and patients may require proportionally high concentrations of stabilizers to achieve long-term protein stability. The resulting formulations may be hypertonic, potentially resulting in injection pain due to tissue damage. Therefore, it is important to balance the amount of stabilizer with the stability and osmolality of the concentrated protein formulation.

[0007] For these reasons, there is a need to develop G-CSF formulations with high protein concentrations without causing protein aggregation, significantly increasing osmotic pressure, and / or significantly decreasing protein stability. Summary of the Invention

[0008] In some embodiments, a pharmaceutical composition is provided that comprises (or consists essentially of, or consists of) (a) about 1 mg / mL to about 100 mg / mL of G-CSF dimer, (b) about 1 mM to about 50 mM of a buffering agent, (c) about 0.1 mM to about 20 mM of a stabilizer, (d) about 1% (w / v) to about 15% (w / v) (e.g., about 1% (w / v) to about 10% (w / v)) of a tonicity agent, and (e) about 0.001% (w / v) to about 0.1% (w / v) of a surfactant.

[0009] In some embodiments of any of the pharmaceutical compositions described above, the G-CSF dimer comprises two monomer subunits, each of which comprises a G-CSF monomer and a dimerization domain. In some embodiments, the G-CSF monomer comprises the sequence of SEQ ID NO: 1. In some embodiments, the G-CSF monomer is connected to the dimerization domain (e.g., within each monomer subunit) via an optional linker. In some embodiments, the linker is about 6 to about 30 amino acids in length. In some embodiments, the linker comprises the sequence of SEQ ID NO: 12. In some embodiments, the dimerization domain comprises at least two cysteines capable of forming intermolecular disulfide bonds. In some embodiments, the dimerization domain comprises at least a portion of an Fc fragment. In some embodiments, the Fc fragment comprises a CH2 and a CH3 domain. In some embodiments, the Fc fragment is derived from IgG1 Fc, IgG2 Fc, IgG4 Fc, or a fragment or variant thereof. In some embodiments, the Fc fragment comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-5. In some embodiments, the G-CSF monomer is N-terminal to the dimerization domain within each monomer subunit. In some embodiments, each monomer subunit comprises an amino acid sequence of any of SEQ ID NOs: 6-10, or a variant thereof having at least about 90% (e.g., at least about 95%) sequence identity to an amino acid sequence of any of SEQ ID NOs: 6-10. In some embodiments, each monomer subunit comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 6-10, e.g., SEQ ID NO: 6.

[0010] In some embodiments of any of the pharmaceutical compositions described above, the G-CSF dimer is efvemarenograstim alfa.

[0011] In some embodiments of any of the pharmaceutical compositions described above, the concentration of the G-CSF dimer is about 5 mg / mL to about 50 mg / mL, for example, about 20 mg / mL.

[0012] In some embodiments of any of the pharmaceutical compositions described above, the buffering agent is sodium acetate, hi some embodiments, the concentration of sodium acetate is about 1 mM to about 30 mM, e.g., about 10 mM.

[0013] In some embodiments of any of the pharmaceutical compositions described above, the stabilizer is EDTA. In some embodiments, the concentration of EDTA is about 0.1 mg / mL to about 10 mg / mL, e.g., about 1 mg / mL.

[0014] In some embodiments of any of the pharmaceutical compositions described above, the tonicity agent is sorbitol or sucrose. In some embodiments, the tonicity agent is sorbitol. In some embodiments, the pharmaceutical composition comprises about 1% (w / v) to about 8% (w / v) sorbitol, e.g., about 5% (w / v) sorbitol. In some embodiments, the tonicity agent is sucrose. In some embodiments, the pharmaceutical composition comprises about 5% (w / v) to about 15% (w / v) sucrose, e.g., about 9% (w / v) sucrose.

[0015] In some embodiments of any of the pharmaceutical compositions described above, the surfactant is selected from the group consisting of polysorbate, poloxamer, polyoxyethylene alkyl ether, alkylphenyl polyoxyethylene ether, and combinations thereof. In some embodiments, the surfactant is polysorbate 20 (PS20) or polysorbate 80 (PS80). In some embodiments, the pharmaceutical composition comprises about 0.005% (w / v) to about 0.05% (w / v) PS20, e.g., about 0.01% (w / v) PS20.

[0016] In some embodiments of any of the pharmaceutical compositions described above, the pH of the pharmaceutical composition is about 4.2 to about 6.2, for example, about 4.8 to about 5.8, about 5.0 to about 5.4, or about 5.2.

[0017] In some embodiments of any of the pharmaceutical compositions described above, the pharmaceutical composition comprises (a) about 20 mg / mL G-CSF dimer, (b) about 10 mM sodium acetate, (c) about 1 mM EDTA, (d) about 5% (w / v) sorbitol, and (e) about 0.01% (w / v) PS20, wherein the pH of the pharmaceutical composition is about 5.2.

[0018] In some embodiments of any of the pharmaceutical compositions described above, the pharmaceutical composition is stable for at least about 1 month at 25±2° C., such as for at least about 2 months at 25±2° C. In some embodiments, the pharmaceutical composition is stable for up to about 3 months at 25±2° C.

[0019] In some embodiments of any of the pharmaceutical compositions described above, the pharmaceutical composition has a purity of at least about 90% G-CSF dimer as assessed by size-exclusion high performance liquid chromatography (SE-HPLC).

[0020] In some embodiments of any of the pharmaceutical compositions described above, the pharmaceutical composition has an osmolality of about 268 mOsm / kg to about 360 mOsm / kg.

[0021] In some embodiments of any of the pharmaceutical compositions described above, the pharmaceutical composition comprises at least about 85% of the main peak relative to the total area of all peaks when measured by SE-HPLC after storage for at least about 1 month (e.g., at least about 2 months) at 25±2° C. In some embodiments, the pharmaceutical composition comprises at least about 85% of the main peak relative to the total area of all peaks when measured by SE-HPLC after storage for at least about 3 months at 25±2° C.

[0022] In some embodiments of any of the pharmaceutical compositions described above, the pharmaceutical composition comprises less than about 2.5% dimer based on the total area of all peaks as measured by SE-HPLC after storage for at least about 1 month (e.g., at least about 2 months) at 25±2° C. In some embodiments, the pharmaceutical composition comprises less than about 2.5% dimer based on the total area of all peaks as measured by SE-HPLC after storage for at least about 3 months at 25±2° C.

[0023] In some embodiments of any of the pharmaceutical compositions described above, the pharmaceutical composition comprises less than about 2% aggregates relative to the total area of all peaks as measured by SE-HPLC after storage for at least about 1 month (e.g., at least about 2 months) at 25±2° C. In some embodiments, the pharmaceutical composition comprises no more than about 2% aggregates relative to the total area of all peaks as measured by SE-HPLC after storage for at least about 3 months at 25±2° C.

[0024] In some embodiments of any of the pharmaceutical compositions described above, the main peak comprises at least about 85% of the total area of all peaks when measured by SE-HPLC after storage for at least about 36 months at about 2° C. to about 8° C. In some embodiments, the pharmaceutical composition comprises at least about 85% of the main peak relative to the total area of all peaks when measured by SE-HPLC after storage for up to about 36 months at about 2° C. to about 8° C.

[0025] In some embodiments of any of the pharmaceutical compositions described above, the pharmaceutical composition comprises at least about 96% G-CSF dimer as measured by reduced capillary electrophoresis-sodium dodecyl sulfate (rCE-SDS) after storage for at least about 1 month (e.g., at least about 2 months) at 25±2° C. In some embodiments, the pharmaceutical composition comprises at least about 96% G-CSF dimer as measured by rCE-SDS after storage for up to about 3 months at 25±2° C.

[0026] In some embodiments of any of the pharmaceutical compositions described above, the pharmaceutical composition comprises at least about 95.5% G-CSF dimer as measured by rCE-SDS after at least about 36 months of storage at about 2° C. to about 8° C. In some embodiments, the pharmaceutical composition comprises at least about 95.5% G-CSF dimer as measured by rCE-SDS after up to about 36 months of storage at about 2° C. to about 8° C.

[0027] In some embodiments of any of the pharmaceutical compositions described above, the pharmaceutical composition comprises about 1.5% or less high molecular weight species (HMWS) as measured by rCE-SDS after storage for at least about 1 month (e.g., at least about 2 months) at 25±2° C. In some embodiments, the pharmaceutical composition comprises about 1.5% or less HMWS as measured by rCE-SDS after storage for up to about 3 months at 25±2° C.

[0028] In some embodiments of any of the pharmaceutical compositions described above, the pharmaceutical composition comprises about 1.5% or less high molecular weight species as measured by rCE-SDS after at least about 36 months of storage at about 2° C. to about 8° C. In some embodiments, the pharmaceutical composition comprises about 1.5% or less HMWS as measured by rCE-SDS after up to about 36 months of storage at about 2° C. to about 8° C.

[0029] In some embodiments of any of the pharmaceutical compositions described above, the pharmaceutical composition comprises at least about 94% G-CSF dimer as measured by non-reducing capillary electrophoresis-sodium dodecyl sulfate (nrCE-SDS) after storage for at least about 1 month (e.g., at least about 2 months) at 25±2° C. In some embodiments, the pharmaceutical composition comprises at least about 94% G-CSF dimer as measured by nrCE-SDS after storage for up to about 3 months at 25±2° C.

[0030] In some embodiments of any of the pharmaceutical compositions described above, the pharmaceutical composition comprises at least about 94% G-CSF dimer as measured by nrCE-SDS after at least about 36 months of storage at about 2° C. to about 8° C. In some embodiments, the pharmaceutical composition comprises at least about 94% G-CSF dimer as measured by nrCE-SDS after up to about 36 months of storage at about 2° C. to about 8° C.

[0031] In some embodiments of any of the pharmaceutical compositions described above, the pharmaceutical composition comprises about 1.5% or less high molecular weight species as measured by nrCE-SDS after storage for at least about 1 month (e.g., at least about 2 months) at 25±2° C. In some embodiments, the pharmaceutical composition comprises about 1.5% or less HMWS as measured by nrCE-SDS after storage for up to about 3 months at 25±2° C.

[0032] In some embodiments of any of the pharmaceutical compositions described above, the pharmaceutical composition comprises no more than about 1.5% high molecular weight species as measured by nrCE-SDS after at least about 36 months of storage at about 2° C. to about 8° C. In some embodiments, the pharmaceutical composition comprises no more than about 1.5% high molecular weight species as measured by nrCE-SDS after up to about 36 months of storage at about 2° C. to about 8° C.

[0033] In some embodiments of any of the pharmaceutical compositions described above, the pharmaceutical composition comprises about 27.2% to about 39.0% acidic area, about 36.5% to about 60.1% main peak, and about 7.2% to about 30% basic area as measured by imaging capillary isoelectric focusing (icIEF) after storage for at least about 1 month (e.g., at least about 2 months) at 25±2° C. In some embodiments, the pharmaceutical composition comprises about 27.2% to about 39.0% acidic area, about 36.5% to about 60.1% main peak, and about 7.2% to about 30% basic area as measured by icIEF after storage for up to about 3 months at 25±2° C.

[0034] In some embodiments of any of the pharmaceutical compositions described above, the pharmaceutical composition comprises about 27.2% to about 39.0% acidic area, about 36.5% to about 60.1% main peak, and about 7.2% to about 30% basic area as measured by icIEF after at least about 36 months of storage at 2° C. to 8° C. In some embodiments, the pharmaceutical composition comprises about 27.2% to about 39.0% acidic area, about 36.5% to about 60.1% main peak, and about 7.2% to about 30% basic area as measured by icIEF after up to about 36 months of storage at about 2° C. to about 8° C.

[0035] In some embodiments of any of the pharmaceutical compositions described above, the pharmaceutical composition retains at least about 70% potency after storage for at least about 1 month (e.g., at least about 2 months) at 25±2° C. In some embodiments, the pharmaceutical composition retains at least about 70% potency after storage for up to about 3 months at 25±2° C.

[0036] In some embodiments of any of the pharmaceutical compositions described above, the pharmaceutical composition retains at least about 70% potency after at least about 36 months of storage at about 2° C. to about 8° C. In some embodiments, the pharmaceutical composition retains at least about 70% potency after up to about 36 months of storage at about 2° C. to about 8° C.

[0037] In some embodiments of any of the pharmaceutical compositions described above, the concentration of the pharmaceutical composition changes by less than about 0.5 mg / mL after storage for at least about 1 month (e.g., at least about 2 months) at 25±2° C. In some embodiments, the concentration of the pharmaceutical composition changes by less than about 0.5 mg / mL after storage for up to about 3 months at 25±2° C.

[0038] In some embodiments of any of the pharmaceutical compositions described above, the concentration of the pharmaceutical composition changes by less than about 0.5 mg / mL after at least about 36 months of storage at about 2° C. to about 8° C. In some embodiments, the concentration of the pharmaceutical composition changes by less than about 0.5 mg / mL after up to about 36 months of storage at about 2° C. to about 8° C.

[0039] In some embodiments of any of the pharmaceutical compositions described above, the pharmaceutical composition is contained in a syringe. In some embodiments, the volume of the pharmaceutical composition in the syringe is about 1 mL. In some embodiments, the syringe is for single use. In some embodiments, the syringe is sterile.

[0040] In another aspect, a syringe containing any of the pharmaceutical compositions described above is provided. In some embodiments, the syringe is for single use. In some embodiments, the volume of the liquid pharmaceutical composition in the syringe is about 1 mL. In some embodiments, the syringe is sterile.

[0041] In a further aspect, methods are provided for treating or preventing a disease or condition in an individual (e.g., a human), comprising administering to the individual an effective amount of any of the pharmaceutical compositions described herein (e.g., a liquid pharmaceutical composition). In some embodiments, the pharmaceutical composition is administered at a dose of about 0.01 mg / kg to about 1 mg / kg. In some embodiments, the pharmaceutical composition is administered once every four weeks. In some embodiments, the pharmaceutical composition is administered once every three weeks. In some embodiments, the pharmaceutical composition is administered subcutaneously. In some embodiments, the disease or condition is selected from the group consisting of neutropenia, stroke, spinal cord injury, neurological disorders associated with blood-brain barrier damage, Parkinson's disease, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, spinal muscular atrophy, primary lateral sclerosis, spinocerebellar ataxia, and mobilization of hematopoietic stem cells into peripheral blood in allogeneic blood stem cell transplantation. In some embodiments, the disease or condition is neutropenia. In some embodiments, the neutropenia is chemotherapy-induced neutropenia or radiation therapy-induced neutropenia. In some embodiments, the method comprises administering an effective amount of a pharmaceutical composition to an individual after administration of a chemotherapeutic agent to the individual. In some embodiments, the chemotherapeutic agent is a myelosuppressive chemotherapeutic agent. In some embodiments, the pharmaceutical composition is administered at about 20 mg / kg once per chemotherapy cycle. In some embodiments, the individual is administered at least four cycles of the chemotherapeutic agent, and the pharmaceutical composition is administered after administration of the chemotherapeutic agent in each of the at least four cycles. In some embodiments, the pharmaceutical composition is administered at about 5 mg to about 25 mg (e.g., about 20 mg) of G-CSF dimer per administration.

[0042] In a further aspect, there is provided a method of producing any of the pharmaceutical compositions disclosed herein, comprising: (i) culturing host cells under conditions suitable for expression of a G-CSF dimer, (ii) isolating the expressed G-CSF dimer from the cell culture, (iii) purifying the expressed G-CSF dimer, and (iv) formulating the purified G-CSF dimer with a buffer, a stabilizer, a tonicity agent, and a surfactant. In some embodiments, the purification step comprises one or more of affinity chromatography, viral inactivation, ion exchange chromatography, mixed-mode chromatography, and filtration. [Brief explanation of the drawings]

[0043] [Figure 1] 1 is an illustration of the structure of an exemplary G-CSF dimer (e.g., efvemarenograstim alfa). In the figure, "-" represents a linker, the oval object labeled "G-CSF" represents a G-CSF monomer, and the oval object labeled "Fc" represents an Fc fragment (e.g., Fc from human IgG). The exemplary G-CSF dimer comprises two monomer subunits. Each monomer subunit comprises, from N' to C', a G-CSF monomer, a linker, and an Fc fragment. [Figure 2] 1 shows an exemplary chromatogram by reversed-phase high-performance liquid chromatography (RP-HPLC) of a G-CSF-Fc dimer pharmaceutical composition containing 15 mg / mL G-CSF-Fc dimer, 10 mM sodium acetate, 5% (w / v) sorbitol, 0.01% (w / v) polysorbate 20, 1 mM EDTA, pH 5.2, after 12 weeks of storage at 25±2° C. Three major elution peaks are present: impurity peak 1, impurity peak 2, and main peak. [Figure 3A-3B](●) 10 mM sodium acetate, 5% (w / v) sorbitol, 1 mM EDTA, 0.01% (w / v) polysorbate 20, pH 4.8, (■) 10 mM sodium acetate, 5% (w / v) sorbitol, 1 mM EDTA, 0.01% (w / v) polysorbate 20, pH 5.2, (▲) 10 mM sodium acetate, 9% (w / v) sucrose, 1 mM E after storage for up to 12 weeks at 25±2°C. 1 shows the change in the percentage (%) of the main band of G-CSF dimer measured by non-reducing SDS-PAGE (A) and reducing SDS-PAGE (B) in a G-CSF dimer pharmaceutical composition containing DTA, 0.01% (w / v) polysorbate 20, pH 4.8, or (×) 10 mM sodium acetate, 9% (w / v) sucrose, 1 mM EDTA, 0.01% (w / v) polysorbate 20, pH 5.2. [Figure 3C-3D] After storage at 25±2°C for up to 12 weeks, (●) 10 mM sodium acetate, 5% (w / v) sorbitol, 1 mM EDTA, 0.01% (w / v) polysorbate 20, pH 4.8, (■) 10 mM sodium acetate, 5% (w / v) sorbitol, 1 mM EDTA, 0.01% (w / v) polysorbate 20, pH 5.2, (▲) 10 mM sodium acetate, 9% (w / v) sucrose, 1 mM EDTA, 0.0 The percentage of the main peak (%) (C) and the percentage of impurity peak 2 (D) versus time measured by RP-HPLC are shown for a G-CSF dimer pharmaceutical composition containing 1% (w / v) polysorbate 20, pH 4.8, or (×) 10 mM sodium acetate, 9% (w / v) sucrose, 1 mM EDTA, 0.01% (w / v) polysorbate 20, pH 5.2. [Figure 4A]The figure shows the change in the percentage of the main band of G-CSF dimer measured by non-reducing SDS-PAGE in G-CSF dimer pharmaceutical compositions containing (●) 10 mM sodium acetate, 5% (w / v) sorbitol, 0.5 mM EDTA, 0.01% (w / v) polysorbate 20, pH 5.2, (■) 10 mM sodium acetate, 5% (w / v) sorbitol, 1 mM EDTA, 0.01% (w / v) polysorbate 20, pH 5.2, or (▲) 10 mM sodium acetate, 5% (w / v) sorbitol, 0.5 mM EDTA, 0.03% (w / v) polysorbate 20, pH 5.2, after storage at 25±2°C for up to 12 weeks. [Figure 4B] The change in the percentage of the main band of G-CSF dimer measured by reducing SDS-PAGE is shown in the graph for G-CSF dimer pharmaceutical compositions containing (●) 10 mM sodium acetate, 5% (w / v) sorbitol, 0.5 mM EDTA, 0.01% (w / v) polysorbate 20, pH 5.2, (■) 10 mM sodium acetate, 5% (w / v) sorbitol, 1 mM EDTA, 0.01% (w / v) polysorbate 20, pH 5.2, or (▲) 10 mM sodium acetate, 5% (w / v) sorbitol, 0.5 mM EDTA, 0.03% (w / v) polysorbate 20, pH 5.2, after storage at 25±2°C for up to 12 weeks. [Figure 4C] The percentage of the main peak over time measured by RP-HPLC is shown for G-CSF dimer pharmaceutical compositions containing (●) 10 mM sodium acetate, 5% (w / v) sorbitol, 0.5 mM EDTA, 0.01% (w / v) polysorbate 20, pH 5.2, (■) 10 mM sodium acetate, 5% (w / v) sorbitol, 1 mM EDTA, 0.01% (w / v) polysorbate 20, pH 5.2, or (▲) 10 mM sodium acetate, 5% (w / v) sorbitol, 0.5 mM EDTA, 0.03% (w / v) polysorbate 20, pH 5.2 after storage at 25±2°C for up to 12 weeks. [Figure 4D]Shown is the percentage of impurity peak 2 versus time as measured by RP-HPLC in G-CSF dimer pharmaceutical compositions containing (●) 10 mM sodium acetate, 5% (w / v) sorbitol, 0.5 mM EDTA, 0.01% (w / v) polysorbate 20, pH 5.2, (■) 10 mM sodium acetate, 5% (w / v) sorbitol, 1 mM EDTA, 0.01% (w / v) polysorbate 20, pH 5.2, or (▲) 10 mM sodium acetate, 5% (w / v) sorbitol, 0.5 mM EDTA, 0.03% (w / v) polysorbate 20, pH 5.2 after storage for up to 12 weeks at 25±2°C. [Figure 4E] (×) 10 mM sodium acetate, 9% (w / v) sucrose, 0.5 mM EDTA, 0.01% (w / v) polysorbate 20, pH 5.2, (◆) 10 mM sodium acetate, 9% (w / v) sucrose, 1 mM EDTA, 0.01% (w / v) polysorbate 20, pH 5.2, or (●) 10 mM sodium acetate, 9% (w / v) sucrose, 1 mM EDTA, 0.01% (w / v) polysorbate 20, pH 5.2, after storage at 25 ± 2°C for up to 12 weeks. 1 shows the change in the percentage of the main band of G-CSF dimer measured by non-reducing SDS-PAGE in a G-CSF dimer pharmaceutical composition containing (○) 10 mM sodium acetate, 9% (w / v) sucrose, 0.5 mM EDTA, 0.03% (w / v) polysorbate 20, pH 5.2, or (○) 10 mM sodium acetate, 9% (w / v) sucrose, 0.5 mM EDTA, 0.03% (w / v) polysorbate 20, pH 5.2. [Figure 4F](×) 10 mM sodium acetate, 9% (w / v) sucrose, 0.5 mM EDTA, 0.01% (w / v) polysorbate 20, pH 5.2, (◆) 10 mM sodium acetate, 9% (w / v) sucrose, 1 mM EDTA, 0.01% (w / v) polysorbate 20, pH 5.2, or (●) 10 mM sodium acetate, 9% (w / v) sucrose, 1 mM EDTA, 0.01% (w / v) polysorbate 20, pH 5.2, after storage at 25 ± 2°C for up to 12 weeks. 1 shows the change in the percentage of the main band of G-CSF dimer measured by reducing SDS-PAGE in a G-CSF dimer pharmaceutical composition containing (○) 10 mM sodium acetate, 9% (w / v) sucrose, 0.5 mM EDTA, 0.03% (w / v) polysorbate 20, pH 5.2, or (○) 10 mM sodium acetate, 9% (w / v) sucrose, 0.5 mM EDTA, 0.03% (w / v) polysorbate 20, pH 5.2. [Figure 4G] Figure 1 shows the percentage of the main peak over time as measured by RP-HPLC for a G-CSF dimer pharmaceutical composition containing (x) 10 mM sodium acetate, 9% (w / v) sucrose, 0.5 mM EDTA, 0.01% (w / v) polysorbate 20, pH 5.2, (◆) 10 mM sodium acetate, 9% (w / v) sucrose, 1 mM EDTA, 0.01% (w / v) polysorbate 20, pH 5.2, or (○) 10 mM sodium acetate, 9% (w / v) sucrose, 0.5 mM EDTA, 0.03% (w / v) polysorbate 20, pH 5.2 after storage at 25 ± 2°C for up to 12 weeks. [Figure 4H] Figure 1 shows the percentage of impurity peak 2 versus time as measured by RP-HPLC in a G-CSF dimer pharmaceutical composition containing (×) 10 mM sodium acetate, 5% (w / v) sucrose, 0.5 mM EDTA, 0.01% (w / v) polysorbate 20, pH 5.2, (◆) 10 mM sodium acetate, 5% (w / v) sucrose, 1 mM EDTA, 0.01% (w / v) polysorbate 20, pH 5.2, or (bal) 10 mM sodium acetate, 5% (w / v) sucrose, 0.5 mM EDTA, 0.03% (w / v) polysorbate 20, pH 5.2 after storage at 25 ± 2°C for up to 12 weeks. [Figure 5]1 shows a chromatogram of size-exclusion high-performance chromatography (SE-HPLC) of a G-CSF-Fc dimer pharmaceutical composition containing 20 mg / mL G-CSF-Fc dimer, 10 mM sodium acetate, 5% (w / v) sorbitol, 0.01% (w / v) polysorbate 20, 1 mM EDTA, pH 5.2 at week 0. Two major elution peaks, namely, the dimer and the main peak, are present. [Figure 6] Shown is the percentage of dimer over time measured by SE-HPLC in G-CSF dimer pharmaceutical compositions containing (●) 10 mM sodium acetate, 5% (w / v) sorbitol, 0.5 mM EDTA, 0.01% (w / v) polysorbate 20, pH 5.2, (■) 10 mM sodium acetate, 5% (w / v) sorbitol, 1 mM EDTA, 0.01% (w / v) polysorbate 20, pH 5.2, or (▲) 10 mM sodium acetate, 5% (w / v) sorbitol, 0.5 mM EDTA, 0.03% (w / v) polysorbate 20, pH 5.2 after storage at 25±2°C for up to 12 weeks. DETAILED DESCRIPTION OF THE INVENTION

[0044] The present application provides G-CSF formulations (eg, pharmaceutical compositions) in liquid or lyophilized form that are stable under suitable storage conditions. The present application is based on the unexpected discovery that formulations comprising suitable excipients and G-CSF molecules, particularly G-CSF dimers (e.g., G-CSF-Fc dimers, e.g., efvemarenograstim alfa), are highly stable upon storage. In some embodiments, the G-CSF formulation comprises at least about 97% (e.g., at least about any of 97%, 98%, 99%, or 100%) G-CSF dimers as measured by size exclusion chromatography (SEC) before and / or after storage. In some embodiments, the G-CSF formulation comprises at least about 95.5% G-CSF dimers as measured by rCE-SDS before and / or after storage (e.g., after at least about 1 month of storage at 25±2°C, or after at least about 36 months of storage at about 2°C to about 8°C). In some embodiments, the G-CSF formulation comprises at least about 95.5% G-CSF dimers as measured by rCE-SDS before and / or after storage (e.g., after at least about 1 month of storage at 25±2°C, or after at least about 36 months of storage at about 2°C to about 8°C). contains at least about 94% (e.g., at least about 95%) G-CSF dimer as measured by nrCE-SDS after storage (e.g., after at least about 1 month of storage at 25±2°C, or after at least about 36 months of storage at about 2°C to about 8°C). In some embodiments, the G-CSF formulation contains about 1.5% or less (e.g., about 1% or less) high molecular weight species (HMWS) as measured by nrCE-SDS before and / or after storage (e.g., after at least about 1 month of storage at 25±2°C, or after at least about 36 months of storage at about 2°C to about 8°C). In some embodiments, the G-CSF formulation contains about 1.5% or less (e.g., about 0.5% or less) HMW species (HMWS) as measured by nrCE-SDS before and / or after storage (e.g., after at least about 1 month of storage at 25±2°C, or after at least about 36 months of storage at about 2°C to about 8°C). In some embodiments, the G-CSF formulation comprises at least about 50% of the main peak as measured by icIEF before and / or after storage (e.g., after at least about 1 month of storage at 25±2°C, or after at least about 36 months of storage at about 2°C to about 8°C).In some embodiments, the G-CSF formulation retains at least about 70% (e.g., at least about 80%, 85%, 90%, 95%, 99%, or more) potency before and / or after storage (e.g., after at least about 1 month of storage at 25±2°C, or after at least about 36 months of storage at about 2°C to about 8°C). In some embodiments, the G-CSF formulation changes by less than about 0.5 mg / mL before and / or after storage (e.g., after at least about 1 month of storage at 25±2°C, or after at least about 36 months of storage at about 2°C to about 8°C). These properties make the G-CSF formulation particularly suitable as a pharmaceutical that can be safely administered to human patients, for example, by subcutaneous administration. In some embodiments, the pharmaceutical compositions described herein provide a high concentration of G-CSF (e.g., G-CSF dimer) in a small volume (e.g., 1 mL) for ease of therapeutic administration or delivery.

[0045] Furthermore, the G-CSF formulations described herein (e.g., G-CSF-Fc dimer formulations) improve in vivo tolerability and, consequently, patient compliance by maintaining the osmolality of the formulation. In some embodiments, the osmolality of the G-CSF formulation is about 268 mOsm / kg to about 360 mOsm / kg, e.g., about 310 mOsm / kg. Furthermore, the formulations described herein allow for convenient processing, including, but not limited to, ultrafiltration and sterile filtration, and injection of the drug product solution via a needle.

[0046] Thus, in one aspect, the present application provides a pharmaceutical composition comprising (or consisting essentially of, or consisting of) (a) about 1 mg / mL to about 100 mg / mL of G-CSF dimer (e.g., G-CSF-Fc dimer, e.g., efvemarenograstim alfa), (b) about 1 mM to about 50 mM of buffer, (c) about 0.1 mM to about 20 mM of stabilizer, (d) about 1% (w / v) to about 15% (w / v) (e.g., about 1% (w / v) to about 10% (w / v)) of an isotonicity agent, and (e) about 0.001% (w / v) to about 0.1% (w / v) of a surfactant. In some embodiments, the pH of the pharmaceutical composition is about 4.2 to about 6.2 (e.g., about 5.0 to about 5.4, e.g., about 5.2). In another aspect, there is provided a method for evaluating the suitability of a pharmaceutical composition for medical use in an individual (e.g., a human individual), the method comprising: (a) about 1 mg / mL to about 100 mg / mL of a G-CSF molecule (e.g., a -CSF-Fc dimer, e.g., efvemarenograstim alfa); (b) about 1 mM to about 50 mM of a buffering agent; (c) about 0.1 mM to about 20 mM of a stabilizer; (d) about 1% (w / v) to about 15% (w / v) (e.g., about 1% (w / v) to about 10% (w / v)) of an isotonicity agent; and (e) about 0.001% of a G-CSF molecule (e.g., a G-CSF-Fc dimer, e.g., efvemarenograstim alfa). and about 0.1% (w / v) to about 0.1% (w / v) surfactant, wherein the pH of the pharmaceutical composition is about 4.2 to about 6.2 (e.g., about 5.0 to about 5.4, e.g., about 5.2), and the method includes measuring a quality control parameter of the pharmaceutical composition and assessing the suitability of the pharmaceutical composition for medical use in an individual (e.g., a human individual), wherein a quality control parameter measured within a quality control parameter threshold indicates the suitability of the pharmaceutical composition for medical use. The quality control parameter can be any one or combination of the parameters described herein for pharmaceutical compositions, e.g., assessed by a corresponding method described herein.

[0047] Also provided are syringes, eg, sterile and / or single-use syringes, containing any of the pharmaceutical compositions described herein (eg, efvemarenograstim alfa pharmaceutical compositions).

[0048] Also provided are methods of treatment using the pharmaceutical compositions described herein, as well as kits and articles of manufacture useful in the methods described herein.

[0049] All publications, including patent documents, scientific articles, and databases, referenced in this application are incorporated in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. To the extent that a definition set forth herein contradicts or otherwise conflicts with a definition set forth in a patent, application, publication, or other publication incorporated herein by reference, the definition set forth herein takes precedence over the definition incorporated herein by reference.

[0050] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0051] I. Definition As used herein, the term "viscosity" refers to the resistance of a liquid formulation to flow, for example, when injected through a syringe needle during administration to a patient. Viscosity measurements can be performed using the cone-and-plate method with a Peltier element set at a defined temperature, such as 5°C to 25°C, as described herein. Typically, a well-defined shear stress gradient is applied to the liquid formulation, and the resulting shear rate is measured. Viscosity is the ratio of shear stress to shear rate. As used herein, viscosity is expressed in units of mPa·S at 5°C to 25°C, where 1 mPa·S = 1 cP. The high-concentration, low-viscosity, substantially isotonic formulations disclosed herein are generally characterized as having a viscosity in the range of 1 to 35 mPa·S at 5°C to 25°C.

[0052] As used herein, the term "osmolality" refers to a measure of solute concentration, defined as the number of millimoles of solute (both non-ionized and ionized forms) per kilogram of solution. The desired level of osmolality can be achieved by adding one or more stabilizers, such as sugars or sugar alcohols, including, but not limited to, mannitol, dextrose, glucose, trehalose, and / or sucrose. Additional stabilizers suitable for imparting osmolality are described in references such as, for example, Handbook of Pharmaceutical Excipients (Fourth Edition, Royal Pharmaceutical Society of Britain, Science & Practice Publishers) or Remington's: The Science and Practice of Pharmacy (Nineteenth Edition, Mack Publishing Company).

[0053] As used herein, the term "about" refers to ±10% of a given unit value. When referring to "about" (a value or parameter) herein, the term includes (and describes) a variation of the value or parameter itself. For example, when referring to "about X," the description includes the description of "X."

[0054] As used herein, the term "about X to Y" has the same meaning as "about X to about Y."

[0055] As used herein, the term "substantially" refers to the qualitative condition of exhibiting a desired characteristic or property to a whole or approximate extent. Those skilled in the art of biology will understand that biological and chemical phenomena rarely, if ever, achieve or avoid absolute results. Thus, the term "substantially" is used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0056] As used herein, the terms "iso-osmotic" and "isotonic" are used interchangeably with the terms "substantially isoosmotic" and "substantially isotonic" and refer to a formulation characterized by having an osmotic pressure that is the same as or at least substantially equal to the osmotic pressure of another solution, achieved by a formulation in which the total concentration of solutes, including permeable and impermeable solutes, in the formulation is the same as or at least substantially equal to the total number of solutes in the other solution. Thus, while one of skill in the art will recognize that "iso-osmotic" and "isotonic" formulations used for in vivo administration generally have an osmolality in the range of about 268 mOsm / kg to about 360 mOsm / kg, in the context of the high concentration, low viscosity formulations of the present embodiments, the terms "iso-osmotic," "isotonic," "substantially isoosmotic," and "substantially isotonic" are used interchangeably to refer to formulations having an osmolality in the range of about 240 mOsm / kg to about 400 mOsm / kg, or about 270 mOsm / kg to about 370 mOsm / kg, or about 300 mOsm / kg to about 330 mOsm / kg.

[0057] As used herein, the term "tonicity agent" or "tonicity excipient" refers to a compound designed to reduce local irritation caused by a pharmaceutical formulation / composition by preventing osmotic shock at the application site. These excipients, which are typically added to injectable, ophthalmic, or intranasal preparations, include, but are not limited to, potassium chloride, sodium chloride, mannitol, sorbitol, dextrose, and glycerin.

[0058] As used herein, the term "buffer" refers to a substance that is useful for maintaining the pH of an aqueous solution within a desired range. Suitable buffers include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some embodiments, a mixture of two or more buffers is used.

[0059] As used herein, the term "stabilizer" refers to a substance useful in maintaining desirable product properties. Exemplary stabilizers include, but are not limited to, amino acids, mannitol, sorbitol, sucrose, trehalose, dextran 40, and any combination thereof.

[0060] As used herein, the term "salt" refers to inorganic salts, including, but not limited to, sodium chloride (NaCl), sodium sulfate (NaSO), sodium thiocyanate (NaSCN), magnesium chloride (MgCl), magnesium sulfate (MgSO), ammonium thiocyanate (NHSCN), ammonium sulfate ((NH)SO), ammonium chloride (NHCl), calcium chloride (CaCl), calcium sulfate (CaSO), zinc chloride (ZnCl), and the like, or combinations thereof.

[0061] As used herein, the term "surfactant" includes non-ionic surfactants, including, but not limited to, polysorbates such as polysorbate 20 or 80, and poloxamers such as poloxamer 184 or 188. The amount of surfactant effective in providing stable, high-concentration G-CSF dimer and other protein formulations typically ranges from 0 ppm to 2000 ppm. The use of non-ionic surfactants allows the formulation to withstand shear and surface stresses without denaturing the G-CSF dimer or other proteins, and also reduces adsorption to surfaces during processing and storage. Formulations disclosed herein include, but are not limited to, formulations with one or more non-surfactants, such as one or more polysorbates, e.g., polysorbate 20 or 80, and one or more poloxamers, e.g., poloxamer 184 or 188. Exemplary formulations herein include formulations with polysorbates, such as polysorbate 20 (Tween® 20) or polysorbate 80 (Tween® 80).

[0062] As used herein, the term "protein" refers to an amino acid polymer comprising at least five constituent amino acids covalently linked by peptide bonds, which can be from the group of amino acids encoded by the genetic code, including alanine, valine, leucine, isoleucine, methionine, phenylalanine, tyrosine, tryptophan, serine, threonine, asparagine, glutamine, cysteine, glycine, proline, arginine, histidine, lysine, aspartic acid, and glutamic acid.

[0063] As used herein, the term "protein" is synonymous with the related terms "peptide" and "polypeptide."

[0064] As used herein, the term "antibody" includes full-length antibodies, diabodies, single-chain Fv (scFv) fragments, Fab fragments, Fab' fragments, F(ab'), Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv), V H Fc-Fc fusions, scFv-Fc fusions, scFv-Fv fusions, diabodies, tribodies, and tetrabodies. "Antibody" may refer to the class of proteins commonly known as immunoglobulins. Antibodies include full-length monoclonal antibodies (mAbs), such as IgG4 monoclonal antibodies, which contain the Fc region of an immunoglobulin.

[0065] The term "immunoglobulin" refers to a class of structurally related glycoproteins consisting of two pairs of polypeptide chains, one pair of low molecular weight light (L) chains and one pair of heavy (H) chains, all four of which are interconnected by disulfide bonds. The structure of immunoglobulins has been extensively characterized. See, e.g., Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, NY (1989)). Briefly, each heavy chain typically comprises a heavy chain variable region (referred to herein as V H ) and the heavy chain constant region (C HThe heavy chain constant region consists of three domains: CH1, CH2, and CH3. Each light chain generally contains a light chain variable region (referred to herein as V L The light chain constant region is generally referred to herein as C L It consists of one domain, abbreviated as V. H and V L The regions can be further subdivided into regions of hypervariability (or hypervariable regions that may be hypervariable in sequence and / or formed by structurally defined loops), also called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). H and V L is typically composed of three CDRs and four FRs arranged from amino to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and Lesk J. Mol. Biol. 196, 901-917 (1987)).

[0066] Unless otherwise specified herein, the numbering of amino acid residues in an IgG Fc region is according to the EU numbering system for antibodies, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0067] The "CH1 domain" (also referred to as the "C1" of the "H1" domain) typically spans from approximately amino acid 118 to approximately amino acid 215 (EU numbering system). The "hinge region" is generally defined as the region of IgG corresponding to Glu216 to Pro230 of human IgG1 (Burton, Molec. Immunol. 22:161-206 (1985)). Hinge regions of other IgG isotypes can be aligned with the IgG1 sequence by placing the first and last cysteine residues that form inter-heavy chain disulfide bonds in the same positions. The "CH2 domain" (also referred to as the "C2" domain) of the human IgG Fc domain typically spans from approximately amino acid 231 to approximately amino acid 340. The CH2 domain is unique in that it is not tightly paired with another domain. Rather, two N-linked branched carbohydrate chains are inserted between the two CH2 domains in intact native IgG molecules. It has been speculated that carbohydrates may provide an alternative to domain-domain pairing and help stabilize the CH2 domain. Burton, Molec Immunol. 22:161-206 (1985). The "CH3 domain" (also referred to as the "C3" domain) comprises the stretch from the C-terminal residue of the Fc domain to the CH2 domain (i.e., from about amino acid residue 341 to the C-terminus of the antibody sequence, typically amino acid residue 446 or 447 for IgG).

[0068] The terms "Fc domain" or "fragment crystallizable region" herein are used to define the C-terminal region of an immunoglobulin heavy chain and include native-sequence Fc domains and variant Fc domains. Although the boundaries of an immunoglobulin heavy chain Fc domain can vary, the Fc domain of a human IgG heavy chain is usually defined as extending from amino acid residue position Cys226 or Pro230 to its carboxyl terminus. The C-terminal lysine of the Fc domain (residue 447 according to the EU numbering system) can be removed, for example, during production or purification of the Fc-containing protein or by genetically engineering a nucleic acid encoding the Fc-containing protein. Thus, a composition of Fc-containing proteins can include an Fc-containing protein population in which all K447 residues have been removed, an Fc-containing protein population in which the K447 residue has not been removed, and an Fc-containing protein population having a mixture of Fc-containing proteins with and without the K447 residue removed. Native sequence Fc domains suitable for use in the Fc-containing proteins described herein may include human IgG1, IgG2 (IgG2A, IgG2B), IgG3 and IgG4.

[0069] "Percent amino acid sequence identity" or "homology" with respect to the polypeptide sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the polypeptide being compared, after aligning the sequences and taking into account any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), or MUSCLE software. Those skilled in the art can determine appropriate parameters for assessing alignment, including any algorithms required to achieve maximum alignment across the entire length of the sequences being compared. However, for purposes herein, percent amino acid sequence identity values are generated using the sequence comparison computer program MUSCLE (Edgar, RC, Nucleic Acids Research 32(5):1792-1797, 2004; Edgar, RC, BMC Bioinformatics 5(1):113, 2004).

[0070] As used herein, the term "pharmaceutically effective amount" of a G-CSF dimeric molecule formulation refers to the amount of the formulation that provides a therapeutic effect in a dosing regimen.

[0071] As used herein, the terms "effective amount," "pharmaceutically effective amount," and "therapeutically effective amount" refer to a non-toxic but sufficient amount of an agent to produce a desired biological result. The result can be a reduction (e.g., at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% reduction) and / or alleviation of the signs, symptoms, or causes of a disease or disorder, or any other desired alteration of a biological system. As will be recognized by one of skill in the art, the effective amount of an agent can vary depending on factors such as the desired biological endpoint, the pharmacokinetics of the compound, the disease being treated, the mode of administration, and the age, weight, health, and condition of the subject.

[0072] As used herein, the term "pharmaceutical formulation" or "pharmaceutical composition" refers to a preparation in which the biological activity of the active ingredient contained therein is effective and which does not contain any ingredient that is unacceptably toxic to the subject to whom the formulation is administered.

[0073] A "sterile" preparation is sterile or free or essentially free of viable microorganisms and their spores.

[0074] As used herein, the term "absolute neutrophil count (ANC)" is a measure of the number of neutrophil granulocytes present in the blood. Neutrophils are a type of white blood cell that play a central role in the immune system's defense against infections, particularly bacterial infections. They are the most abundant type of white blood cell in most mammals and form an integral part of the innate immune system.

[0075] As used herein, "myelosuppression" refers to the suppression of one or more components of hematopoiesis, manifested as abnormal levels of one or more of the cell types that are the product of this process. A review of hematopoiesis and the characteristics of hematopoietic cells can be found in Clinical Immunology: Principes and Practice, Vol. 1, Ch. 2, pp. 15-24 (Lewis and Harriman, eds. Mosby-Year Edition, Inc. 1996), which pages are incorporated herein by reference. At a general level, myelosuppression refers to a decrease in white blood cell (WBC) and / or platelet counts. At a more specific level, it also refers to the suppression of one or more of the following cells resulting from hematopoiesis: B cells, T cells, natural killer cells, dendritic cells, macrophages, neutrophils, eosinophils, basophils, mast cells, and platelets. In contrast, "bone marrow recovery" is therefore the opposite of myelosuppression. A "myelosuppressive agent" or "myelosuppressive therapy" is a drug or treatment that is capable of inducing myelosuppression.

[0076] As used herein, the term "neutropenia" is defined as a condition characterized by a decreased number of neutrophils. Normal is an absolute neutrophil count of 2.0 x 10 9 Grade 1 is defined as an absolute neutrophil count of 1.5 × 10 9 / L~1.999×10 9 Grade 2 is defined as neutropenia with an absolute neutrophil count of 1.0 × 10 / L. 9 / L~1.499×10 9 Grade 3 is defined as neutropenia with an absolute neutrophil count of 0.5 × 10 / L. 9 / L~0.999×10 9 Grade 4 (severe neutropenia) is defined as an absolute neutrophil count of 0.5 × 10 9 / L. The terms "severe neutropenia" and "grade 4 neutropenia" may be used interchangeably.

[0077] The term "fever neutropenia" (FN) is defined as a single oral temperature of 38.3°C (101°F) or higher or a temperature of 38.0°C (100.4°F) or higher sustained for 1 hour, accompanied by neutropenia, and is usually classified as an ANC of less than 500 cells / mm3 or an ANC predicted to decrease to less than 500 cells / mm3 over the next 48 hours. It is a common and serious complication in people undergoing cancer treatment, especially chemotherapy.

[0078] "Subjects" to which administration is contemplated include, but are not limited to, humans (i.e., male or female of any age, e.g., a pediatric subject (e.g., infant, child, adolescent) or an adult subject (e.g., young adult, middle-aged adult, or elderly adult)) and / or non-human animals, e.g., mammals such as primates (e.g., cynomolgus monkeys, rhesus monkeys), cows, pigs, horses, sheep, goats, rodents, cats, and / or dogs. In some embodiments, the individual is a human. In certain embodiments, the individual is a non-human animal. The individual can be of any age and / or sex. In some embodiments, the individual has cancer.

[0079] The terms "disease," "disorder," and "condition" are used interchangeably herein.

[0080] As used herein, "treatment" or "treating" is an approach to obtaining beneficial or desired results, including clinical results. For purposes of this application, beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms attributable to the disease (e.g., fever, infection, decreased ANC), reducing the extent of the disease (e.g., lowering the grade of neutropenia), reducing the duration of the disease, stabilizing the disease (e.g., preventing or delaying worsening of the disease), preventing or delaying recurrence of the disease, delaying or slowing disease progression (e.g., progression to the next grade of neutropenia or febrile neutropenia), improving the disease state, causing remission (partial or complete) of the disease, reducing the dose of one or more other medications required to treat the disease, delaying disease progression, increasing or improving quality of life, increasing weight gain, and / or prolonging survival. "Treatment" also encompasses the reduction of pathological consequences (e.g., fever, infection, decreased ANC), including the severity and incidence of pathological consequences. The methods of the present application contemplate any one or more of these aspects of treatment. Where the method is for treating cancer, the method in some embodiments may also prevent or slow the spread of cancer (e.g., metastasis) and / or reduce pathological consequences (e.g., tumor volume).

[0081] As used herein, "reference" refers to any sample, standard, or level used for comparison purposes. The reference can be obtained from a healthy sample and / or a non-disease sample. In some examples, the reference can be obtained from an untreated sample. In some examples, the reference is obtained from a non-disease or untreated sample of an individual. In some examples, the reference is obtained from one or more healthy individuals who are not individuals or patients.

[0082] As used herein, "delaying the onset of disease" means to postpone, prevent, slow, retard, stabilize, inhibit, and / or postpone the onset of the disease. This delay can be of varying lengths of time, depending on the history of the disease and / or the individual being treated. As will be apparent to one of skill in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease.

[0083] "Preventing" as used herein includes providing protection against the onset or recurrence of a disease in an individual who may have a predisposition to the disease, but who has not yet been diagnosed with the disease.

[0084] It will be understood that embodiments of the invention described herein include "consisting of" and / or "consisting essentially of" embodiments.

[0085] As used herein, "not" a value or parameter generally means and describes "other than" a value or parameter. For example, a method is not used to treat cancer of type X means that the method is used to treat cancers of types other than X.

[0086] As used in this specification and the appended claims, the singular forms "a," "or," and "the" include plural referents unless the context clearly dictates otherwise.

[0087] II. Pharmaceutical Compositions In some embodiments, a pharmaceutical composition is provided comprising a G-CSF molecule. Any of the G-CSF molecules described herein, including but not limited to a G-CSF dimer (e.g., any of the G-CSF dimers described herein), can be used in the pharmaceutical composition. In some embodiments, the pharmaceutical composition comprises (or consists essentially of, or consists of) (a) about 1 mg / mL to about 100 mg / mL of a G-CSF molecule (e.g., a G-CSF dimer, e.g., a G-CSF-Fc dimer); (b) about 1 mM to about 50 mM of a buffering agent (e.g., sodium acetate); (c) about 0.1 mM to about 20 mM of a stabilizer (e.g., EDTA); (d) about 1% (w / v) to about 15% (w / v) (e.g., about 1% (w / v) to about 10% (w / v)) of a tonicity agent (e.g., sorbitol or sucrose); and (e) about 0.001% (w / v) to about 0.1% (w / v) of a surfactant (e.g., polysorbate 20 or polysorbate 80). In some embodiments, the pH of the pharmaceutical composition is about 4.2 to about 6.2, e.g., about 5.0 to about 5.4, or about 5.2. In some embodiments, the pharmaceutical composition is contained in a pre-filled syringe. In some embodiments, the volume of the pharmaceutical composition in the syringe is about 1 mL. In some embodiments, the syringe is for single use. In some embodiments, the syringe is sterile. In some embodiments, the G-CSF dimer comprises two monomer subunits, each comprising (or consisting essentially of, or consisting of) the amino acid sequence of any of SEQ ID NOS: 6-10 (e.g., SEQ ID NO: 6). In some embodiments, the G-CSF dimer is efvemarenograstim alfa.

[0088] In some embodiments, provided herein is a pharmaceutical composition comprising a G-CSF dimer, such as any of the G-CSF dimers described herein. In some embodiments, the G-CSF dimer comprises two monomer subunits, each of which comprises a G-CSF monomer and a dimerization domain. In some embodiments, the G-CSF dimer has a long serum half-life. In some embodiments, each monomer subunit comprises (or consists essentially of, or consists of) the amino acid sequence of SEQ ID NO: 6. In some embodiments, the G-CSF dimer is efvemarenograstim alfa.

[0089] In some embodiments, the pharmaceutical composition comprises about 1 mM to about 50 mM of a buffering agent (e.g., sodium acetate). In some embodiments, the pharmaceutical composition comprises about 0.1 mM to about 20 mM of a stabilizer (e.g., EDTA). In some embodiments, the pharmaceutical composition comprises about 1% (w / v) to about 15% (w / v), e.g., about 1% (w / v) to about 10% (w / v), of an isotonicity agent (e.g., sorbitol or sucrose). In some embodiments, the pharmaceutical composition comprises about 0.001% (w / v) to about 0.1% (w / v) of a surfactant (e.g., polysorbate 20). In some embodiments, the pH of the pharmaceutical composition is about 4.2 to about 6.2, e.g., about 4.8 to about 5.8, about 5.0 to about 5.4, or about 5.2.

[0090] Typically, the G-CSF dimer in the pharmaceutical composition remains soluble at a concentration of about 1 mg / mL to about 100 mg / mL and is stable under isotonic storage conditions, demonstrating improved stability compared to currently available G-CSF formulations. In some embodiments, the pharmaceutical composition contains about 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 10 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, or 95 mg / mL or more of the G-CSF dimer. In some embodiments, the pharmaceutical composition contains about 1 mg / mL to about 100 mg / mL of G-CSF dimer, e.g., about 1 mg / mL to about 90 mg / mL, about 1 mg / mL to about 80 mg / mL, about 1 mg / mL to about 70 mg / mL, about 1 mg / mL to about 60 mg / mL, about 1 mg / mL to about 50 mg / mL, about 2 mg / mL to about 50 mg / mL, about 5 mg / mL to about 50 mg / mL, about 5 mg / mL to about 40 mg / mL In some embodiments, the pharmaceutical composition contains about 18 mg / mL to about 22 mg / mL of G-CSF dimer, e.g., about 20 mg / mL of G-CSF dimer.

[0091] Buffering agents include, but are not limited to, citrate, phosphate, acetate, succinate, tartrate, maleate, HEPES, Tris, bicine, glycine, N-glycylglycine, carbonate, glycylglycine, lysine, arginine, histidine, and / or mixtures thereof. In some embodiments, the buffering agent is sodium acetate. In some embodiments, the pharmaceutical composition contains about 1 mM to about 50 mM sodium acetate, for example, about 1 mM to about 40 mM, about 1 mM to about 30 mM, about 5 mM to about 25 mM, about 5 mM to about 20 mM, or about 10 mM sodium acetate.

[0092] In some embodiments, in the pharmaceutical compositions described herein, sodium acetate is a buffer that can be used to maintain the pH of the pharmaceutical composition at about pH 4.2 to about pH 6.2, e.g., about pH 4.4 to about pH 6.0, about pH 4.6 to about pH 5.8, about pH 4.8 to about pH 5.8, about pH 4.8 to about pH 5.6, or about pH 5.0 to about pH 5.4. In some embodiments, the pH of the pharmaceutical composition is about pH 5.0 to about pH 5.4 (pH 5.2±0.2), e.g., about pH 5.2.

[0093] Stabilizers can be added to improve the stability and shelf life of the pharmaceutical compositions described herein. Examples of stabilizers include, but are not limited to, glycine, alanine, glutamic acid, methionine, arginine, benzoic acid, citric acid, glycolic acid, lactic acid, malic acid, maleic acid, polyols (such as sorbitol, mannitol, and trehalose), surfactants, ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), hydroxyethylenediaminetriacetic acid (HEDTA), ethyleneglycol-bis-(2-aminoethyl)-N,N,N',N'-tetraacetic acid (EGTA), nitrilotriacetic acid (NTA), metal ion stabilizers, and citrate salts. In some embodiments, the stabilizer is EDTA. In some embodiments, the pharmaceutical composition comprises about 0.1 mM to about 20 mM EDTA, e.g., about 0.1 mM to about 15 mM, about 0.1 mM to about 10 mM, about 0.5 mM to about 5 mM, about 0.5 mM to about 2 mM, about 1 mM to about 2 mM, or about 1 mM EDTA. EDTA may function as a chelating agent, preservative, or stabilizer to prevent catalytic oxidation reactions in the pharmaceutical composition during processing and storage.

[0094] In some embodiments, the pharmaceutical composition comprises one or more tonicity adjusting agents. The tonicity adjusting agents reduce local irritation caused by the pharmaceutical formulation / composition by preventing osmotic shock at the application site (e.g., by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%). Examples of tonicity adjusting agents include, but are not limited to, sugar alcohols or polyols (such as mannitol or polyols), nonionic surfactants (such as polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80), and sugars (such as sucrose, maltose, trehalose, etc.). In some embodiments, the tonicity adjusting agent is sorbitol. In some embodiments, the pharmaceutical composition comprises about 1% (w / v) to about 15% (w / v) sorbitol, e.g., about 1% (w / v) to about 10% (w / v), about 2% (w / v) to about 10% (w / v), about 3% (w / v) to about 10% (w / v), about 4% (w / v) to about 10% (w / v), about 5% (w / v) to about 10% (w / v), about 5% (w / v) to about 9% (w / v), about 5% (w / v) to about 8% (w / v), about 5% (w / v) to about 7% (w / v), about 5% (w / v) to about 6% (w / v), or about 5% (w / v) sorbitol. In some embodiments, the tonicity agent is sucrose. In some embodiments, the pharmaceutical composition contains about 1% (w / v) to about 15% (w / v) sucrose, for example, about 5% (w / v) to about 15% (w / v), 5% (w / v) to about 10% (w / v), about 6% (w / v) to about 15% (w / v), about 7% (w / v) to about 15% (w / v), about 8% (w / v) to about 15% (w / v), about 9% (w / v) to about 15% (w / v), or about 9% (w / v) to about 14% (w / v). % (w / v), about 9% (w / v) to about 13% (w / v), about 9% (w / v) to about 12% (w / v), about 9% (w / v) to about 11% (w / v), about 9% (w / v) to about 10% (w / v), or about 9% (w / v) of sucrose.

[0095] Nonionic surfactants such as polysorbates, including Polysorbate (Tween®) 20 and Polysorbate (Tween®) 80; poloxamers, including Poloxamers 184 and 188; Plronic® polyols, and other ethylene / polypropylene block polymers stabilize formulations during processing and storage by reducing interfacial interactions and preventing protein adsorption. In some embodiments, the surfactant is selected from the group consisting of polysorbates, poloxamers, polyoxyethylene alkyl ethers, alkylphenyl polyoxyethylene ethers, and combinations thereof. In some embodiments, the surfactant is Polysorbate 20 (PS20) or Polysorbate 80 (PS80). In some embodiments, the pharmaceutical composition comprises Polysorbate 20 (PS20). In some embodiments, the pharmaceutical composition comprises about 0.001% (w / v) to about 0.1% (w / v) polysorbate 20, for example, about 0.001% (w / v) to about 0.09% (w / v), about 0.001% (w / v) to about 0.08% (w / v), about 0.001% (w / v) to about 0.07% (w / v), about 0.001% (w / v) to about 0.06% (w / v), about 0.001% (w / v) to about 0.05% (w / v), about Contains any of 0.001% (w / v) to about 0.04% (w / v), about 0.001% (w / v) to about 0.03% (w / v), about 0.001% (w / v) to about 0.02% (w / v), about 0.001% (w / v) to about 0.01% (w / v), about 0.005% (w / v) to about 0.05% (w / v), about 0.008% (w / v) to about 0.012% (w / v), or about 0.01% (w / v) of polysorbate 20.

[0096] Exemplary herein are G-CSF dimer pharmaceutical compositions in which the G-CSF dimer is soluble at high protein concentrations and stable upon long-term storage.

[0097] In some embodiments, the pharmaceutical composition comprises: (a) about 1 mg / mL to about 100 mg / mL (e.g., about 5 mg / mL to about 50 mg / mL, e.g., about 20 mg / mL, etc.) of G-CSF dimer; (b) about 1 mM to about 50 mM (e.g., about 1 mM to about 30 mM, e.g., about 10 mM) of a buffering agent (e.g., sodium acetate); (c) about 0.1 mM to about 20 mM (e.g., about 0.1 mM to about 10 mM, e.g., about 1 mM) of a stabilizer (e.g., EDTA); and (d) about 1% (w / v) to about 15% (w / v) (e.g., about 1% (w / v) to about 10% (w / v), e.g., about 5% (w / v) or about 9% (w / v)) of a soluble G-CSF dimer. and (e) about 0.001% (w / v) to about 0.1% (w / v) (e.g., about 0.005% (w / v) to about 0.05% (w / v), e.g., about 0.01% (w / v)) of a surfactant (e.g., polysorbate 20 or polysorbate 80), the pharmaceutical composition comprising (or consisting essentially of, or consisting of) the following: (a) a tonicity agent (e.g., sorbitol or sucrose); and (b) about 0.001% (w / v) to about 0.1% (w / v) (e.g., about 0.005% (w / v) to about 0.05% (w / v), e.g., about 0.01% (w / v)) of a surfactant (e.g., polysorbate 20 or polysorbate 80); the pharmaceutical composition has a pH of about 4.2 to about 6.2 (e.g., about 5.0 to about 5.4, or about 5.2); and the G-CSF dimer comprises two monomer subunits, each monomer subunit comprising a G-CSF monomer and a dimerization domain.In some embodiments, the pharmaceutical composition comprises: (a) about 1 mg / mL to about 100 mg / mL (e.g., about 5 mg / mL to about 50 mg / mL), (e.g., about 20 mg / mL) of G-CSF dimer; (b) about 1 mM to about 50 mM (e.g., about 1 mM to about 30 mM, e.g., about 10 mM) of a buffering agent (e.g., sodium acetate); (c) about 0.1 mM to about 20 mM (e.g., about 0.1 mM to about 10 mM, e.g., about 1 mM) of a stabilizer (e.g., EDTA); and (d) about 1% (w / v) to about 15% (w / v) (e.g., about 1% (w / v) to about 10% (w / v), e.g., about 5% (w / v)) or about 9% (w / v)) of an isotonicity agent (e.g., sorbitol or and (e) about 0.001% (w / v) to about 0.1% (w / v) (e.g., about 0.005% (w / v) to about 0.05% (w / v), e.g., about 0.01% (w / v)) of a surfactant (e.g., polysorbate 20 or polysorbate 80), the pH is about 4.2 to about 6.2 (e.g., about 5.0 to about 5.4, or about 5.2), the G-CSF dimer comprises two monomer subunits, each monomer subunit comprising a G-CSF monomer, an Fc fragment, and an optional linker connecting the G-CSF monomer and the Fc fragment. In some embodiments, the G-CSF monomer comprises the sequence of SEQ ID NO: 1. In some embodiments, the G-CSF monomer is connected to the dimerization domain via an optional linker, such as a linker comprising the amino acid sequence of SEQ ID NO: 12. In some embodiments, the dimerization domain comprises at least a portion of an Fc fragment. In some embodiments, the Fc fragment comprises CH2 and CH3 domains. In some embodiments, the Fc fragment is derived from IgG1 Fc, IgG2 Fc, IgG4 Fc, or fragments or variants thereof. In some embodiments, the Fc fragment comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-5. In some embodiments, the G-CSF monomer is N-terminal to the dimerization domain in each monomer subunit.In some embodiments, each monomeric subunit comprises (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 6-10, or a variant thereof having at least about 90% sequence identity to the amino acid sequence of any of SEQ ID NOs: 6-10. In some embodiments, the pharmaceutical composition comprises: (a) about 1 mg / mL to about 100 mg / mL (e.g., about 5 mg / mL to about 50 mg / mL), (e.g., about 20 mg / mL) of G-CSF dimer; (b) about 1 mM to about 50 mM (e.g., about 1 mM to about 30 mM, e.g., about 10 mM) of a buffering agent (e.g., sodium acetate); (c) about 0.1 mM to about 20 mM (e.g., about 0.1 mM to about 10 mM, e.g., about 1 mM) of a stabilizer (e.g., EDTA); and (d) about 1% (w / v) to about 15% (w / v) (e.g., about 1% (w / v) to about 10% (w / v), e.g., about 5% (w / v)) or about 9% (w / v)) of an isotonicity agent (e.g., sorbitol or and (e) about 0.001% (w / v) to about 0.1% (w / v) (e.g., about 0.005% (w / v) to about 0.05% (w / v), e.g., about 0.01% (w / v)) of a surfactant (e.g., polysorbate 20 or polysorbate 80), the pH is about 4.2 to about 6.2 (e.g., about 5.0 to about 5.4, or about 5.2), and the G-CSF dimer comprises two monomer subunits each comprising (or consisting essentially of, or consisting of) the amino acid sequence of any of SEQ ID NOs: 6 to 10 (e.g., SEQ ID NO: 6).In some embodiments, the pharmaceutical composition comprises: (a) about 1 mg / mL to about 100 mg / mL (e.g., about 5 mg / mL to about 50 mg / mL), (e.g., about 20 mg / mL) of G-CSF dimer; (b) about 1 mM to about 50 mM (e.g., about 1 mM to about 30 mM, e.g., about 10 mM) of sodium acetate; (c) about 0.1 mM to about 20 mM (e.g., about 0.1 mM to about 10 mM, e.g., about 1 mM) of EDTA; (d) about 1% (w / v) to about 15% (w / v) (e.g., about 1% (w / v) to about 10% (w / v), e.g., about 5% (w / v)) or about 9% (w / v)) of sorbitol; and (e) about 0.001% and a surfactant (e.g., polysorbate 20 or polysorbate 80) at a concentration of from about 0.005% (w / v) to about 0.05% (w / v), e.g., about 0.01% (w / v)), the pH is from about 4.2 to about 6.2 (e.g., from about 5.0 to about 5.4, or about 5.2), and the G-CSF dimer comprises two monomer subunits each comprising (or consisting essentially of, or consisting of) the amino acid sequence of any of SEQ ID NOs: 6 to 10 (e.g., SEQ ID NO: 6).In some embodiments, the pharmaceutical composition comprises: (a) about 1 mg / mL to about 100 mg / mL (e.g., about 5 mg / mL to about 50 mg / mL), (e.g., about 20 mg / mL) of G-CSF dimer; (b) about 1 mM to about 50 mM (e.g., about 1 mM to about 30 mM, e.g., about 10 mM) of sodium acetate; (c) about 0.1 mM to about 20 mM (e.g., about 0.1 mM to about 10 mM, e.g., about 1 mM) of EDTA; (d) about 1% (w / v) to about 15% (w / v) (e.g., about 5% (w / v) to about 15% (w / v), e.g., about 9% (w / v)) of sucrose; and (e) about 0.001% (w / v) to about 15% (w / v) of sucrose. and about 0.1% (w / v) (e.g., about 0.005% (w / v) to about 0.05% (w / v), e.g., about 0.01% (w / v)), a surfactant (e.g., polysorbate 20 or polysorbate 80), and the pH is about 4.2 to about 6.2 (e.g., about 5.0 to about 5.4, or about 5.2), and the G-CSF dimer comprises two monomer subunits each comprising (or consisting essentially of, or consisting of) the amino acid sequence of any of SEQ ID NOs: 6 to 10 (e.g., SEQ ID NO: 6). In some embodiments, a pharmaceutical composition is provided comprising (or consisting essentially of, or consisting of) (a) about 20 mg / mL G-CSF dimer, (b) about 10 mM sodium acetate, (c) about 1 mM EDTA, (d) about 5% (w / v) sorbitol, and (e) about 0.01% (w / v) polysorbate 20; the pH is about 5.0 to about 5.4 (e.g., about 5.2); and the G-CSF dimer comprises two monomeric subunits, each comprising (or consisting essentially of, or consisting of) the amino acid sequence of any of SEQ ID NOS: 6-10 (e.g., SEQ ID NO: 6). In some embodiments, the G-CSF dimer is efvemarenograstim alfa. In some embodiments, the pharmaceutical composition is packaged in a pre-filled syringe. In some embodiments, the volume of the pharmaceutical composition in the syringe is about 1 mL. In some embodiments, the syringe is single use. In some embodiments, the syringe is sterile.

[0098] In some embodiments, the pharmaceutical composition includes: (a) a G-CSF dimer (e.g., a G-CSF-Fc dimer, e.g., efvemarenograstim alfa); (b) about 1 mM to about 50 mM (e.g., about 1 mM to about 30 mM, e.g., about 10 mM) of a buffering agent (e.g., sodium acetate); (c) about 0.1 mM to about 20 mM (e.g., about 0.1 mM to about 10 mM, e.g., about 1 mM) of a stabilizer (e.g., EDTA); and (d) about 1% (w / v) to about 15% (w / v) (e.g., about 1% (w / v) to about 10 (e) about 0.001% (w / v) to about 0.1% (w / v) (e.g., about 0.005% (w / v) to about 0.05% (w / v), e.g., about 0.01% (w / v)) of a surfactant (e.g., polysorbate 20 or polysorbate 80), and having a pH of about 4.2 to about 6.2 (e.g., about 5.0 to about 5.4, In some embodiments, a pharmaceutical composition is provided, the pharmaceutical composition comprising: (a) a G-CSF dimer (e.g., a G-CSF-Fc dimer, e.g., efvemarenograstim alfa); (b) about 1 mM to about 50 mM (e.g., about 1 mM to about 30 mM, e.g., about 10 mM) sodium acetate; (c) about 0.1 mM to about 20 mM (e.g., about 0.1 mM to about 10 mM, e.g., about 1 mM) EDTA; and (d) about 1% (w / v) to about 15% (w / v) (e.g., about 1% (w / v) and (e) about 0.001% (w / v) to about 0.1% (w / v) (e.g., about 0.005% (w / v) to about 0.05% (w / v), e.g., about 0.01% (w / v)) of polysorbate 20 or polysorbate 80 (e.g., polysorbate 20), and the pharmaceutical composition has a pH of about 4.2 to about 6.2 (e.g., about 5.0 to about 5.4, or about 5.2).In some embodiments, the pharmaceutical composition comprises: (a) a G-CSF dimer (e.g., a G-CSF-Fc dimer, e.g., efvemarenograstim alfa); (b) about 1 mM to about 50 mM (e.g., about 1 mM to about 30 mM, e.g., about 10 mM) sodium acetate; (c) about 0.1 mM to about 20 mM (e.g., about 0.1 mM to about 10 mM, e.g., about 1 mM) EDTA; and (d) (e) about 1% (w / v) to about 15% (w / v) (e.g., about 1% (w / v) to about 10% (w / v), e.g., about 9% (w / v)) sucrose, and (e) about 0.001% (w / v) to about 0.1% (w / v) (e.g., about 0.005% (w / v) to about 0.05% (w / v), e.g., about 0.01% (w / v)) polysorbate 20 or polysorbate 80 (e.g., polysorbate 20), and a pH of about 4.2 to about 6.2 (e.g., about 5.0 to about 5.4, or about 5.2). In some embodiments, the G-CSF dimer comprises two monomeric subunits, each comprising (or consisting essentially of, or consisting of) the amino acid sequence of any of SEQ ID NOS: 6-10 (e.g., SEQ ID NO: 6). In some embodiments, the G-CSF dimer is efvemarenograstim alfa. In some embodiments, the pharmaceutical composition is contained in a pre-filled syringe. In some embodiments, the volume of the pharmaceutical composition in the syringe is about 1 mL. In some embodiments, the syringe is for single use. In some embodiments, the syringe is sterile.

[0099] In some embodiments, a pharmaceutical composition is provided comprising (or consisting essentially of, or consisting of) (a) a G-CSF dimer (e.g., a G-CSF-Fc dimer, e.g., efvemarenograstim alfa), (b) about 1 mM to about 50 mM sodium acetate, (c) about 0.1 mM to about 20 mM EDTA, (d) about 1% (w / v) to about 10% (w / v) sorbitol, and (e) about 0.001% (w / v) to about 0.1% (w / v) polysorbate 20. In some embodiments, a pharmaceutical composition is provided comprising (a) about 1 mg / mL to about 100 mg

[0013] Pharmaceutical compositions are provided that comprise (or consist essentially of, or consist of) (a) a G-CSF dimer (e.g., a G-CSF-Fc dimer, e.g., efvemarenograstim alfa) at about 1000 mg / mL (e.g., about 20 mg / mL), (b) about 1 mM to about 50 mM sodium acetate, (c) about 0.1 mM to about 20 mM EDTA, (d) about 1% (w / v) to about 10% (w / v) sorbitol, and (e) about 0.001% (w / v) to about 0.1% (w / v) polysorbate 20. In some embodiments, pharmaceutical compositions are provided that comprise (or consist essentially of, or consist of) (a) a G-CSF dimer (e.g., a G-CSF-Fc dimer, e.g., efvemarenograstim alfa ... For example, a pharmaceutical composition is provided that comprises (or consists essentially of, or consists of) (a) about 1 mg / mL to about 100 mg / mL (e.g., about 20 mg / mL) of G-CSF-Fc dimer (e.g., efvemarenograstim alfa), (b) about 1 mM to about 50 mM sodium acetate, (c) about 0.1 mM to about 20 mM EDTA, (d) about 1% (w / v) to about 15% (w / v) sucrose, and (e) about 0.001% (w / v) to about 0.1% (w / v) polysorbate 20. In some embodiments, the pharmaceutical composition comprises (a) about 1 mg / mL to about 100 mg / mL (e.g., about 20 mg / mL) of G-CSF dimer (e.g., efvemarenograstim alfa), (b) about 1 mM to about 50 mM sodium acetate, (c) about 0.1 mM to about 20 mM EDTA, (d) about 1% (w / v) to about 15% (w / v) sucrose, and (e) about 0.001% (w / v) to about 0.1% (w / v) polysorbate 20. For example, provided is a pharmaceutical composition comprising (or consisting essentially of, or consisting of) a G-CSF-Fc dimer (e.g., efvemarenograstim alfa), (b) about 1 mM to about 50 mM sodium acetate, (c) about 0.1 mM to about 20 mM EDTA, (d) about 1% (w / v) to about 15% (w / v) sucrose, and (e) about 0.001% (w / v) to about 0.1% (w / v) polysorbate 20. In some embodiments, the pH of the pharmaceutical composition is about 4.2 to about 6.2, e.g., about 5.0 to about 5.4, or about 5.2.In some embodiments, the G-CSF dimer comprises two monomeric subunits, each comprising (or consisting essentially of, or consisting of) the amino acid sequence of any of SEQ ID NOS: 6-10 (e.g., SEQ ID NO: 6). In some embodiments, the G-CSF dimer is efvemarenograstim alfa. In some embodiments, the pharmaceutical composition is contained in a pre-filled syringe. In some embodiments, the volume of the pharmaceutical composition in the syringe is about 1 mL. In some embodiments, the syringe is for single use. In some embodiments, the syringe is sterile.

[0100] In some embodiments, the composition comprises (or consists essentially of, or consists of): (a) a G-CSF dimer (e.g., a G-CSF-Fc dimer, e.g., efvemarenograstim alfa); (b) about 1 mM to about 50 mM sodium acetate; (c) about 0.1 mM to about 20 mM EDTA; (d) about 1% (w / v) to about 10% (w / v) sorbitol; and (e) about 0.001% (w / v) to about 0.1% (w / v) polysorbate 20; Pharmaceutical compositions are provided having a pH of about 4.2 to about 6.2 (e.g., about 5.0 to about 5.4, or about 5.2). In some embodiments, the pharmaceutical compositions comprise: (a) about 1 mg / mL to about 100 mg / mL (e.g., about 18 mg / mL to about 22 mg / mL, or about 20 mg / mL) of G-CSF dimer (e.g., G-CSF-Fc dimer, e.g., efvemarenograstim alfa); (b) about 1 mM to about 50 mM sodium acetate; and (c) about 0.1 mM to about 20 mM E. Pharmaceutical compositions are provided that comprise (or consist essentially of, or consist of) DTA, (d) about 1% (w / v) to about 10% (w / v) sorbitol, and (e) about 0.001% (w / v) to about 0.1% (w / v) polysorbate 20, and have a pH of about 4.2 to about 6.2 (e.g., about 5.0 to about 5.4, or about 5.2). In some embodiments, the pharmaceutical compositions include (a) a G-CSF dimer (e.g., a G-CSF-Fc dimer, e ... (b) about 1 mM to about 50 mM sodium acetate; (c) about 0.1 mM to about 20 mM EDTA; (d) about 1% (w / v) to about 15% (w / v) sucrose; and (e) about 0.001% (w / v) to about 0.1% (w / v) polysorbate 20, and the pharmaceutical composition has a pH of about 4.2 to about 6.2 (e.g., about 5.0 to about 5.4, or about 5.2).In some embodiments, the solution comprises (or consists essentially of, or consists of) (a) about 1 mg / mL to about 100 mg / mL (e.g., about 18 mg / mL to about 22 mg / mL, or about 20 mg / mL) of a G-CSF dimer (e.g., a G-CSF-Fc dimer, e.g., efvemarenograstim alfa), (b) about 1 mM to about 50 mM sodium acetate, (c) about 0.1 mM to about 20 mM EDTA, (d) about 1% (w / v) to about 15% (w / v) sucrose, and (e) about 0.001% (w / v) to about 0.1% (w / v) polysorbate 20, and has a pH of about 4.2 to about 6.2 (e.g., about 5.0 to about 5.4, or about 5.2). In some embodiments, the G-CSF dimer comprises two monomeric subunits, each comprising (or consisting essentially of, or consisting of) the amino acid sequence of any of SEQ ID NOS: 6-10 (e.g., SEQ ID NO: 6). In some embodiments, the G-CSF dimer is efvemarenograstim alfa. In some embodiments, the pharmaceutical composition is contained in a pre-filled syringe. In some embodiments, the volume of the pharmaceutical composition in the syringe is about 1 mL. In some embodiments, the syringe is for single use. In some embodiments, the syringe is sterile.

[0101] In some embodiments, the solution comprises (or consists essentially of), or is a mixture of (a) a G-CSF dimer (e.g., a G-CSF-Fc dimer, e.g., efvemarenograstim alfa), (b) about 1 mM to about 30 mM sodium acetate, (c) about 0.1 mM to about 10 mM EDTA, (d) about 1% (w / v) to about 8% (w / v) sorbitol, and (e) about 0.005% (w / v) to about 0.05% (w / v) polysorbate 20. and a pH of about 4.8 to about 5.8 (e.g., about 5.0 to about 5.4, or about 5.2). In some embodiments, a pharmaceutical composition is provided, comprising: (a) about 5 mg / mL to about 50 mg / mL (e.g., about 20 mg / mL) of G-CSF dimer (e.g., G-CSF-Fc dimer, e.g., efvemarenograstim alfa); (b) about 1 mM to about 30 mM sodium acetate; (c) about 0.1 mM to about 10 mM EDTA; and (d) about 1% Pharmaceutical compositions are provided that comprise (or consist essentially of, or consist of) (a) about 8% (w / v) to about 8% (w / v) sorbitol, and (b) about 0.005% (w / v) to about 0.05% (w / v) polysorbate 20, and have a pH of about 4.8 to about 5.8 (e.g., about 5.0 to about 5.4, or about 5.2). In some embodiments, the pharmaceutical compositions include (a) a G-CSF dimer (e.g., a G-CSF-Fc dimer, e.g., efvemarenograstim a), (b) a G-CSF-Fc dimer (e.g., a G-CSF-Fc dimer, e.g., efvemarenograstim a), and (c) a G-CSF-Fc dimer (e.g., a G-CSF-Fc dimer, e.g., efvemarenograstim a). (b) about 1 mM to about 30 mM sodium acetate; (c) about 0.1 mM to about 10 mM EDTA; (d) about 5% (w / v) to about 15% (w / v) sucrose; and (e) about 0.005% (w / v) to about 0.05% (w / v) polysorbate 20, and the pharmaceutical composition has a pH of about 4.8 to about 5.8 (e.g., about 5.0 to about 5.4, or about 5.2).In some embodiments, the solution comprises (or consists essentially of, or consists of) (a) about 5 mg / mL to about 50 mg / mL (e.g., about 20 mg / mL) of G-CSF dimer (e.g., G-CSF-Fc dimer, e.g., efvemarenograstim alfa), (b) about 1 mM to about 30 mM sodium acetate, (c) about 0.1 mM to about 10 mM EDTA, (d) about 5% (w / v) to about 15% (w / v) sucrose, and (e) about 0.005% (w / v) to about 0.05% (w / v) polysorbate 20, and has a pH of about 4.8 to about 5.8 (e.g., about 5.0 to about 5.4, or about 5.2). Pharmaceutical compositions are provided. In some embodiments, the G-CSF dimer comprises two monomer subunits, each comprising (or consisting essentially of, or consisting of) the amino acid sequence of any of SEQ ID NOS: 6-10 (e.g., SEQ ID NO: 6). In some embodiments, the G-CSF dimer is efvemarenograstim alfa. In some embodiments, the pharmaceutical composition is contained in a pre-filled syringe. In some embodiments, the volume of the pharmaceutical composition in the syringe is about 1 mL. In some embodiments, the syringe is for single use. In some embodiments, the syringe is sterile.

[0102] In some embodiments, a pharmaceutical composition is provided that comprises (or consists essentially of, or consists of) (a) a G-CSF dimer (e.g., a G-CSF-Fc dimer, e.g., efvemarenograstim alfa), (b) about 10 mM sodium acetate, (c) about 1 mM EDTA, (d) about 5% (w / v) sorbitol, and (e) about 0.01% (w / v) polysorbate 20, and has a pH of about 4.2 to about 6.2 (e.g., about 5.0 to about 5.4, or about 5.2). In some embodiments, pharmaceutical compositions are provided that comprise (or consist essentially of, or consist of) (a) about 20 mM G-CSF dimer (e.g., G-CSF-Fc dimer, e.g., efvemarenograstim alfa), (b) about 10 mM sodium acetate, (c) about 1 mM EDTA, (d) about 5% (w / v) sorbitol, and (e) about 0.01% (w / v) polysorbate 20, and have a pH of about 4.2 to about 6.2 (e.g., about 5.0 to about 5.4, or about 5.2). In some embodiments, a pharmaceutical composition is provided that comprises (or consists essentially of, or consists of) (a) a G-CSF dimer (e.g., a G-CSF-Fc dimer, e.g., efvemarenograstim alfa), (b) about 10 mM sodium acetate, (c) about 1 mM EDTA, (d) about 9% (w / v) sucrose, and (e) about 0.01% (w / v) polysorbate 20, and has a pH of about 4.2 to about 6.2 (e.g., about 5.0 to about 5.4, or about 5.2). In some embodiments, a pharmaceutical composition is provided that comprises (or consists essentially of, or consists of) (a) about 20 mM G-CSF dimer (e.g., a G-CSF-Fc dimer, e.g., efvemarenograstim alfa), (b) about 10 mM sodium acetate, (c) about 1 mM EDTA, (d) about 9% (w / v) sucrose, and (e) about 0.01% (w / v) polysorbate 20, and has a pH of about 4.2 to about 6.2 (e.g., about 5.0 to about 5.4, or about 5.2).In some embodiments, the G-CSF dimer comprises two monomeric subunits, each comprising (or consisting essentially of, or consisting of) the amino acid sequence of any of SEQ ID NOS: 6-10 (e.g., SEQ ID NO: 6). In some embodiments, the G-CSF dimer is efvemarenograstim alfa. In some embodiments, the pharmaceutical composition is contained in a pre-filled syringe. In some embodiments, the volume of the pharmaceutical composition in the syringe is about 1 mL. In some embodiments, the syringe is for single use. In some embodiments, the syringe is sterile.

[0103] In some embodiments, a pharmaceutical composition is provided that comprises (or consists essentially of, or consists of) (a) a G-CSF dimer (e.g., a G-CSF-Fc dimer, e.g., efvemarenograstim alfa), (b) about 10 mM sodium acetate, (c) about 1 mM EDTA, (d) about 9% (w / v) sucrose, and (e) about 0.01% (w / v) polysorbate 20, and has a pH of about 5.0 to about 5.4 (e.g., about 5.2). In some embodiments, a pharmaceutical composition is provided that comprises (or consists essentially of, or consists of) (a) about 20 mM G-CSF dimer (e.g., a G-CSF-Fc dimer, e.g., efvemarenograstim alfa), (b) about 10 mM sodium acetate, (c) about 1 mM EDTA, (d) about 9% (w / v) sucrose, and (e) about 0.01% (w / v) polysorbate 20, and has a pH of about 5.0 to about 5.4 (e.g., about 5.2). Pharmaceutical compositions are provided that comprise (or consist essentially of, or consist of) a G-CSF-Fc dimer (e.g., efvemarenograstim alfa), (b) about 10 mM sodium acetate, (c) about 1 mM EDTA, (d) about 9% (w / v) sucrose, and (e) about 0.01% (w / v) polysorbate 20, and have a pH of about 5.0 to about 5.4 (e.g., about 5.2). In some embodiments, the G-CSF dimers each comprise (or consist of) the amino acid sequence of any of SEQ ID NOS: 6-10 (e.g., SEQ ID NO: 6). In some embodiments, a pharmaceutical composition is provided that comprises (or consists essentially of, or consists of) (a) efvemarenograstim alfa, (b) about 10 mM sodium acetate, (c) about 1 mM EDTA, (d) about 9% (w / v) sucrose, and (e) about 0.01% (w / v) polysorbate 20, and has a pH of about 5.0 to about 5.4 (e.g., about 5.2). In some embodiments, a pharmaceutical composition is provided that comprises (or consists essentially of, or consists of) (a) about 20 mM efvemarenograstim alfa, (b) about 10 mM sodium acetate, (c) about 1 mM EDTA, (d) about 9% (w / v) sucrose, and (e) about 0.01% (w / v) polysorbate 20, and has a pH of about 5.0 to about 5.4 (e.g., about 5.2). (b) about 10 mM sodium acetate; (c) about 1 mM EDTA; (d) about 9% (w / v) sucrose; and (e) about 0.01% (w / v) polysorbate 20, and the pharmaceutical composition has a pH of about 5.0 to about 5.4 (e.g., about 5.2). In some embodiments, the pharmaceutical composition is contained in a pre-filled syringe. In some embodiments, the volume of the pharmaceutical composition in the syringe is about 1 mL. In some embodiments, the syringe is for single use.In some embodiments, the syringe is sterile.

[0104] In some embodiments, a pharmaceutical composition is provided that comprises (or consists essentially of, or consists of) (a) a G-CSF dimer (e.g., a G-CSF-Fc dimer, e.g., efvemarenograstim alfa), (b) about 10 mM sodium acetate, (c) about 1 mM EDTA, (d) about 5% (w / v) sorbitol, and (e) about 0.01% (w / v) polysorbate 20, and has a pH of about 5.0 to about 5.4 (e.g., about 5.2). In some embodiments, a pharmaceutical composition is provided that comprises (or consists essentially of, or consists of) (a) about 20 mM G-CSF dimer (e.g., a G-CSF-Fc dimer, e.g., efvemarenograstim alfa), (b) about 10 mM sodium acetate, (c) about 1 mM EDTA, (d) about 5% (w / v) sorbitol, and (e) about 0.01% (w / v) polysorbate 20, and has a pH of about 5.0 to about 5.4 (e.g., about 5.2). Pharmaceutical compositions are provided that comprise (or consist essentially of, or consist of) a G-CSF-Fc dimer (e.g., efvemarenograstim alfa), (b) about 10 mM sodium acetate, (c) about 1 mM EDTA, (d) about 5% (w / v) sorbitol, and (e) about 0.01% (w / v) polysorbate 20, and have a pH of about 5.0 to about 5.4 (e.g., about 5.2). In some embodiments, the G-CSF dimers each comprise (or are derived from) the amino acid sequence of any of SEQ ID NOS: 6-10 (e.g., SEQ ID NO: 6). In some embodiments, a pharmaceutical composition is provided that comprises (or consists essentially of, or consists of) (a) efvemarenograstim alfa, (b) about 10 mM sodium acetate, (c) about 1 mM EDTA, (d) about 5% (w / v) sorbitol, and (e) about 0.01% (w / v) polysorbate 20, and has a pH of about 5.0 to about 5.4 (e.g., about 5.2). In some embodiments, a pharmaceutical composition is provided that comprises (or consists essentially of, or consists of) (a) about 20 mM efvemarenograstim alfa, (b) about 10 mM sodium acetate, (c) about 1 mM EDTA, (d) about 5% (w / v) sorbitol, and (e) about 0.01% (w / v) polysorbate 20, and has a pH of about 5.0 to about 5.4 (e.g., about 5.2). (b) about 10 mM sodium acetate; (c) about 1 mM EDTA; (d) about 5% (w / v) sorbitol; and (e) about 0.01% (w / v) polysorbate 20, and the pharmaceutical composition has a pH of about 5.0 to about 5.4 (e.g., about 5.2). In some embodiments, the pharmaceutical composition is contained in a pre-filled syringe. In some embodiments, the volume of the pharmaceutical composition in the syringe is about 1 mL. In some embodiments, the syringe is for single use.In some embodiments, the syringe is sterile.

[0105] In some embodiments, a pharmaceutical composition is provided comprising (or consisting essentially of, or consisting of) (a) about 20 mM efvemarenograstim alfa, (b) about 10 mM sodium acetate, (c) about 1 mM EDTA, (d) about 5% (w / v) sorbitol, and (e) about 0.01% (w / v) polysorbate 20, and having a pH of about 5.2. Such a pharmaceutical composition is also referred to herein as an "efvemarenograstim alfa pharmaceutical composition." In some embodiments, a pharmaceutical composition is provided comprising (or consisting essentially of, or consisting of) (a) 20 mM efvemarenograstim alfa, (b) 10 mM sodium acetate, (c) 1 mM EDTA, (d) 5% (w / v) sorbitol, and (e) 0.01% (w / v) polysorbate 20, and having a pH of 5.2. In some embodiments, the pharmaceutical composition is contained in a prefilled syringe. In some embodiments, the volume of the pharmaceutical composition in the syringe is about 1 mL. In some embodiments, the syringe is for single use. In some embodiments, the syringe is sterile. In some embodiments, the pharmaceutical composition is a clear, colorless, preservative-free solution containing 20 mg of efvemarenograstim alfa provided in a prefilled, single-dose syringe with a 27-gauge, ½-inch needle and an UltraSafe Passive™ needle guard. In some embodiments, each syringe contains about 20 mg of efvemarenograstim alfa in a sterile, clear, colorless, preservative-free solution (pH 5.2) comprising acetate (0.6 mg), EDTA (about 0.29 mg), polysorbate 20 (about 0.1 mg), sodium (about 0.23 mg), and sorbitol (about 50 mg) in water for injection. In some embodiments, each syringe contains 20 mg of efvemarenograstim alfa in a sterile, clear, colorless, preservative-free solution (pH 5.2) comprising acetate (0.6 mg), EDTA (0.29 mg), polysorbate 20 (0.1 mg), sodium (0.23 mg), and sorbitol (50 mg) in water for injection.

[0106] In some embodiments, a pharmaceutical composition is provided that comprises (or consists essentially of, or consists of) (a) a G-CSF dimer (e.g., a G-CSF-Fc dimer, e.g., efvemarenograstim alfa), (b) sodium, (c) acetate, (d) EDTA, (e) sorbitol, and (f) polysorbate 20 or polysorbate 80 (e.g., polysorbate 20). ... In some embodiments, pharmaceutical compositions are provided that comprise (or consist essentially of, or consist of) (a) G-CSF dimer (e.g., G-CSF-Fc dimer, e.g., efvemarenograstim alfa), (b) sodium acetate, (c) EDTA, (d) sorbitol, and (e) polysorbate 20 or polysorbate 80 (e.g., polysorbate 20). ... Provided are pharmaceutical compositions comprising (or consisting essentially of, or consisting of) polysorbate 80 (e.g., polysorbate 20). In some embodiments, the pharmaceutical composition comprises (or consists essentially of, or consists of) (a) a G-CSF dimer (e.g., a G-CSF-Fc dimer, e.g., efvemarenograstim alfa), (b) sodium acetate, (c) EDTA, (d) sucrose, and (e) polysorbate 20 or polysorbate 80 (e.g., polysorbate 20). In some embodiments, the pharmaceutical composition has a pH of about 4.2 to about 6.2 (e.g., about 5.0 to about 5.4, or about 5.2). In some embodiments, the pharmaceutical composition further comprises water. In some embodiments, the G-CSF dimer comprises two monomer subunits, each comprising (or consisting essentially of, or consisting of) the amino acid sequence of any of SEQ ID NOS: 6 to 10 (e.g., SEQ ID NO: 6). In some embodiments, the G-CSF dimer is efvemarenograstim alfa.In some embodiments, the pharmaceutical composition comprises (or consists essentially of, or consists of) about 20 mg efvemarenograstim alfa, about 0.6 mg acetate, about 0.29 mg EDTA, about 0.1 mg polysorbate 20, about 0.23 mg sodium, and about 50 mg sorbitol. In some embodiments, the pharmaceutical composition is lyophilized. In some embodiments, the pharmaceutical composition is about 1 mL of solution (e.g., aqueous solution). In some embodiments, the pH of the pharmaceutical composition is about 5.2.

[0107] In some embodiments, a syringe containing any of the pharmaceutical compositions described herein (e.g., a G-CSF-Fc dimer pharmaceutical composition, e.g., an efvemarenograstim alfa pharmaceutical composition) is also provided. In some embodiments, the volume of the pharmaceutical composition in the syringe is about 1 mL. In some embodiments, the syringe is for single use. In some embodiments, the syringe is sterile. In some embodiments, the prefilled syringe is ungraded and is intended only for administering the entire contents of the syringe (20 mg / 1 mL) directly to an individual (e.g., an adult). In some embodiments, the needle cap of the prefilled syringe comprises natural rubber latex.

[0108] Characteristics of the pharmaceutical composition In some embodiments, the pharmaceutical composition has a purity of G-CSF dimer (e.g., G-CSF-Fc dimer, e.g., efvemarenograstim alfa) of at least about 90% (e.g., at least about 91%, 92%, and any of 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) as assessed by SE-HPLC. In some embodiments, purity is measured before storage. In some embodiments, purity is measured after storage. In some embodiments, the storage conditions include storage at about 2°C to about 8°C for at least about 36 months. In some embodiments, the storage conditions include storage at 25±2°C for at least about 1 month (e.g., at least about 2 months). In some embodiments, the storage conditions include storage at about 2°C to about 8°C for up to about 36 months. In some embodiments, the storage conditions include storage at 25±2°C for up to about 3 months.

[0109] The pharmaceutical compositions described herein are characterized in some embodiments by their long-term stability. In some embodiments, pharmaceutical compositions comprising a G-CSF dimer (e.g., a G-CSF-Fc dimer, e.g., efvemarenograstim alfa) are stable at about 2°C to about 8°C for at least about 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 12 months, 18 months, 24 months, 30 months, 36 months, 48 months, 54 months, 60 months, or more. In some embodiments, pharmaceutical compositions comprising a G-CSF dimer (e.g., a G-CSF-Fc dimer, e.g., efvemarenograstim alfa) are stable at about 2°C to about 8°C for up to about 36 months. In some embodiments, the pharmaceutical composition is stable at 25±2° C. for at least about any of 24 hours, 48 hours, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or more. In some embodiments, the pharmaceutical composition is stable at 25±2° C. for up to about 6 months, e.g., up to about 3 months (e.g., stable after 3, 2, or 1 month(s) of storage). In some embodiments, stability is assessed during storage of a liquid pharmaceutical composition under appropriate storage conditions. In some embodiments, stability is assessed during storage of a lyophilized pharmaceutical composition under appropriate storage conditions, with a liquid pharmaceutical composition reconstituted from a lyophilized pharmaceutical composition. In some embodiments, stability is assessed during storage of a frozen pharmaceutical composition under appropriate storage conditions, with a liquid pharmaceutical composition reconstituted from a frozen pharmaceutical composition.

[0110] In some embodiments, the storage conditions include storage at about 2°C to about 8°C. In some embodiments, the storage conditions include storage at 25±2°C. In some embodiments, the storage conditions include storage for at least about 5 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 24 hours, 48 hours, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 12 months, 18 months, 24 months, 30 months, 36 months, 48 months, 54 months, 60 months, or more. In some embodiments, the storage conditions include storage for up to about 36 months, e.g., up to about 6 months, or up to about 3 months. In some embodiments, the storage conditions include storage at about 2°C to about 8°C for up to about 36 months. In some embodiments, the storage conditions include storage at 25±2°C for up to about 3 months. In some embodiments, the storage conditions include protection from light. In some embodiments, the storage conditions include avoiding shaking. In some embodiments, the storage conditions include avoiding freezing. In some embodiments, the storage conditions include freezing and then thawing at about 2°C to about 8°C prior to use. In some embodiments, the storage conditions include avoiding multiple freezing. In some embodiments, the storage conditions include storing at 25±2°C for no more than about 48 hours.

[0111] In some embodiments, the pharmaceutical composition (e.g., a G-CSF-Fc dimer pharmaceutical composition, e.g., an efvemarenograstim alfa pharmaceutical composition) has excellent freeze-thaw stability, e.g., is stable after at least six (e.g., six, seven, eight, or more) cycles of freezing at about -20°C and thawing at room temperature.

[0112] In some embodiments, the pharmaceutical composition (e.g., a G-CSF-Fc dimer pharmaceutical composition, e.g., an efvemarenograstim alfa pharmaceutical composition) is within a clinically acceptable range after storage. In some embodiments, the pharmaceutical composition contains at least about 96% (e.g., at least about 97%, 98%, 99%, or 100%) G-CSF dimer as measured by size exclusion chromatography (SEC) after storage. In some embodiments, the pharmaceutical composition has a purity of at least about 95.5% (e.g., at least about 96%, 97%, 98%, 99%, or 100%) as measured by rCE-SDS after storage. In some embodiments, the pharmaceutical composition has a purity of at least about 94% (e.g., at least about 95%, 96%, 97%, 98%, 99%, or 100%) as measured by nrCE-SDS after storage. In some embodiments, the pharmaceutical composition has an HMWS of about 1.5% or less (e.g., about any of 1.4%, 1.3%, 1.2%, 1.1%, 1.0%, 0.8%, 0.6%, 0.4%, 0.1%, or less) as measured by rCE-SDS after storage. In some embodiments, the pharmaceutical composition has an HMWS of about 1.5% or less (e.g., about any of 1.4%, 1.3%, 1.2%, 1.1%, 1.0%, 0.8%, 0.6%, 0.4%, 0.1%, or less) as measured by nrCE-SDS after storage. In some embodiments, the pharmaceutical composition comprises about 27.2% to about 39.0% (e.g., about 30% to about 40%, e.g., about 32% to about 37%) of acidic region, about 36.5% to about 60.1% (e.g., about 45% to about 55%, e.g., about 47% to about 55%) of main peak, and about 7.2% to about 30% (e.g., about 10% to about 20%, e.g., about 11% to 18%) of basic region, as measured by icIEF after storage. In some embodiments, the pharmaceutical composition retains at least about 70% (e.g., at least about 75%, 80%, 85%, 90%, 95%, 99%, or 100%) of potency (e.g., G-CSF potency) after storage.In some embodiments, the pharmaceutical composition retains about 70% to about 130% (e.g., at least about any of 85%, 90%, 95%, 100%, 110%, 120%, or 130%) potency (e.g., G-CSF potency) after storage, e.g., by a cell proliferation assay. In some embodiments, the change in concentration (e.g., G-CSF-Fc dimer concentration) of the pharmaceutical composition after storage is less than about 0.5 mg / mL (e.g., less than about any of 0.4 mg / mL, 0.3 mg / mL, 0.2 mg / mL, 0.1 mg / mL, 0.05 mg / mL, or less). In some embodiments, the storage conditions include storage at about 2°C to about 8°C for at least about 36 months. In some embodiments, the storage conditions include storage at 25±2°C for at least about 1 month (e.g., at least about 2 months). In some embodiments, the storage conditions include storage at about 2°C to about 8°C for up to about 36 months. In some embodiments, storage conditions include storage at 25±2° C. for up to about 3 months.

[0113] In some embodiments, the efficacy of a G-CSF molecule (e.g., a G-CSF dimer such as a G-CSF-Fc dimer) or a pharmaceutical composition thereof is determined by: i) promoting the proliferation and / or differentiation of blood cells (e.g., neutrophils) (e.g., by at least about 10%, 20%, 50%, 1-fold, 2-fold, 10-fold, 20-fold, or more); ii) enhancing the immune response (e.g., defense against bacterial infection) (e.g., by at least about any of 10%, 20%, 50%, 1-fold, 2-fold, 10-fold, 20-fold, or more); iii) activating mature neutrophils (e.g., by activating mature neutrophils involved in the immune response or synergizing with other hematopoietic growth factors such as stem cell factor, Flt-3 ligand, and GM-CSF). iv) improved hematopoietic function (e.g., by at least about 10%, 20%, 50%, 1-fold, 2-fold, 10-fold, 20-fold, or more); v) increased neutrophil count (e.g., by at least about 10%, 20%, 50%, 1-fold, 2-fold, 10-fold, 20-fold, or more); vi) enhanced neutrophil recruitment (e.g., by at least about 10%, 20%, 50%, 1-fold, 2-fold, 10-fold, 20-fold, or more); and vii) reduced peripheral blood progenitor cell recruitment (e.g., by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%).

[0114] In some embodiments, the pharmaceutical composition (e.g., a liquid pharmaceutical composition) has an appearance having one or more of the following characteristics: i) clear; ii) colorless to slightly yellowish; and iii) essentially free of visible particulates (e.g., about 10% or less, e.g., about 8%, 5%, 2%, 1%, or less). This appearance may be before and / or after storage. In some embodiments, the storage conditions include storage at about 2°C to about 8°C for up to about 36 months. In some embodiments, the storage conditions include storage at 25±2°C for up to about 3 months. In some embodiments, the pharmaceutical composition is placed in a colorimetric cuvette and visually inspected for visible particles against black and white backgrounds, respectively. In some embodiments, the pharmaceutical composition is compared to a colorimetric standard against a white background to assess color.

[0115] In some embodiments, the pharmaceutical composition is characterized by fewer and / or smaller particles. In some embodiments, the pharmaceutical composition contains no more than 6,000 particles per vial having a diameter of 10 μm or greater and no more than 600 particles per vial having a diameter of 25 μm or greater. In some embodiments, the pharmaceutical composition is transferred to a vial and the particles in the vial are examined using a particle size measurement system (e.g., ACCUSize® 780 SIS). The particulate characteristics of the pharmaceutical composition can be measured before and / or after storage. In some embodiments, the storage conditions include storage at about 2° C. to about 8° C. for up to about 36 months. In some embodiments, the storage conditions include storage at 25±2° C. for up to about 3 months.

[0116] In some embodiments, the pharmaceutical compositions described herein (e.g., G-CSF-Fc dimer pharmaceutical compositions, e.g., efvemarenograstim alfa pharmaceutical compositions) are characterized by low viscosity. In some embodiments, the viscosity of the pharmaceutical composition ranges from about 1 to about 50 mPa·S at about 5°C to about 25°C, e.g., from about 1 to about 45 mPa·S, from about 1 to about 40 mPa·S, from about 1 to about 35 mPa·S, from about 1 to about 30 mPa·S, from about 1 to about 25 mPa·S, or from about 1 to about 20 mPa·S, from about 1 to about 15 mPa·S, from about 1 to about 10 mPa·S, from about 1 to about 5 mPa·S, from about 1 to about 4 mPa·S, from about 1 to about 3 mPa·S, or from about 1 to about 2 mPa·S. For example, in some embodiments, the pharmaceutical composition has a viscosity of about 1 to 2 mPa·S at about 25°C. In some embodiments, the pharmaceutical composition is isotonic. The viscosity characteristics of the pharmaceutical composition may be before and / or after storage. In some embodiments, storage conditions include storage at about 2°C to about 8°C for up to about 36 months. In some embodiments, storage conditions include storage at 25±2°C for up to about 3 months.

[0117] In some embodiments, the osmolality of a pharmaceutical composition described herein (e.g., a G-CSF-Fc dimer pharmaceutical composition such as an efvemarenograstim alfa pharmaceutical composition) is in the range of about 100 to about 500 mOsm / kg, e.g., about 120 to about 480 mOsm / kg, about 140 to about 440 mOsm / kg, about 160 to about 420 mOsm / kg, about 180 to about 400 mOsm / kg, about 200 to about 380 mOsm / kg, about 220 to about 360 mOsm / kg, about 270 to about 350 mOsm / kg, or about 268 to about 360 mOsm / kg. In some embodiments, the osmolality of the pharmaceutical composition is about 310 mOsm / kg. The osmolality characteristics of the pharmaceutical composition may be measured before and / or after storage. In some embodiments, the storage conditions include storage at about 2° C. to about 8° C. for up to about 36 months. In some embodiments, the storage conditions include storage at 25±2° C. for up to about 3 months.

[0118] In some embodiments, a pharmaceutical composition described herein (e.g., a G-CSF-Fc dimer pharmaceutical composition, e.g., an efvemarenograstim alfa pharmaceutical composition) is characterized by a low percent of protein aggregation (e.g., before and / or after storage). In some embodiments, the percent of total protein aggregation is less than about 5%, e.g., less than about any of 4%, 3%, 2%, 1%, 0.5%, 0.2%, 0.1%, or less. In some embodiments, the percent of total protein aggregation is less than about 2% as measured by SEC. In some embodiments, the storage conditions include storage at about 2°C to about 8°C for at least about 36 months. In some embodiments, the storage conditions include storage at 25±2°C for at least about 1 month (e.g., at least about 2 months). In some embodiments, the storage conditions include storage at about 2°C to about 8°C for up to about 36 months. In some embodiments, the storage conditions include storage at 25±2°C for up to about 3 months.

[0119] In some embodiments, the pharmaceutical composition (e.g., a G-CSF-Fc dimer pharmaceutical composition, e.g., an efvemarenograstim alfa pharmaceutical composition) comprises about 1.5% or less (e.g., about 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.8%, 0.5%, 0.1%, or less, e.g., about 1% or less) HMWS as measured by rCE-SDS after storage at 25±2° C. for at least about 1 month. In some embodiments, the pharmaceutical formulation comprises about 1.5% or less (e.g., about 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.8%, 0.5%, 0.1%, or less, e.g., about 1% or less) HMWS as measured by rCE-SDS after storage at 25±2° C. for at least about 2 months. In some embodiments, the pharmaceutical formulation comprises about 1.5% or less HMWS as measured by rCE-SDS after up to about 3 months of storage at 25±2° C. In some embodiments, the pharmaceutical composition comprises about 1.5% or less (e.g., about 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.8%, 0.5%, 0.1%, or less) HMWS as measured by rCE-SDS after at least about 36 months of storage at about 2° C. to about 8° C. In some embodiments, the pharmaceutical composition comprises about 1.5% or less HMWS as measured by rCE-SDS after up to about 36 months of storage at about 2° C. to about 8° C. In some embodiments, the pharmaceutical composition comprises about 1.5% or less (e.g., about 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.8%, 0.5%, 0.1%, or less) HMWS as measured by nrCE-SDS after at least about 1 month of storage at 25±2° C. In some embodiments, the pharmaceutical formulation comprises about 1.5% or less (e.g., about 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.8%, 0.5%, 0.1%, or less) HMWS as measured by nrCE-SDS after at least about 2 months of storage at 25±2° C. In some embodiments, the pharmaceutical formulation comprises about 1.5% or less HMWS as measured by nrCE-SDS after up to about 3 months of storage at 25±2° C.In some embodiments, the pharmaceutical composition comprises about 1.5% or less (e.g., about 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.8%, 0.5%, 0.1%, or less) HMWS as measured by nrCE-SDS after at least about 36 months of storage at about 2° C. to about 8° C. In some embodiments, the pharmaceutical composition comprises about 1.5% or less HMWS as measured by nrCE-SDS after up to about 36 months of storage at about 2° C. to about 8° C.

[0120] In some embodiments, the pharmaceutical composition comprises at least about 90% (e.g., at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) G-CSF dimer (e.g., a G-CSF-Fc dimer pharmaceutical composition, e.g., an efvemarenograstim alfa pharmaceutical composition) after storage. In some embodiments, the pharmaceutical composition comprises at least about 90% (e.g., at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) G-CSF dimer, e.g., at least about 95.5% or at least about 96% G-CSF dimer, as measured by rCE-SDS after at least about 1 month of storage at 25±2°C. In some embodiments, the pharmaceutical composition comprises at least about 90% (e.g., at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) G-CSF dimer, e.g., at least about 95.5% or at least about 96% G-CSF dimer, as measured by rCE-SDS after storage at 25±2° C. for at least about 2 months. In some embodiments, the pharmaceutical composition comprises at least about 90% G-CSF dimer, e.g., at least about 95.5% or at least about 96% G-CSF dimer, as measured by rCE-SDS after storage at 25±2° C. for up to about 3 months. In some embodiments, the pharmaceutical composition comprises at least about 90% (e.g., at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) G-CSF dimer, e.g., at least about 95.5% G-CSF dimer, as measured by rCE-SDS after at least about 36 months of storage at about 2° C. to about 8° C. In some embodiments, the pharmaceutical composition comprises at least about 90% G-CSF dimer, e.g., at least about 95.5% G-CSF dimer, as measured by rCE-SDS after up to about 36 months of storage at about 2° C. to about 8° C.In some embodiments, the pharmaceutical composition comprises at least about 90% (e.g., at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) G-CSF dimer, e.g., at least about 94% G-CSF dimer, as measured by nrCE-SDS after at least about 1 month of storage at 25±2° C. In some embodiments, the pharmaceutical composition comprises at least about 90% (e.g., at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) G-CSF dimer, e.g., at least about 94% G-CSF dimer, as measured by nrCE-SDS after at least about 2 months of storage at 25±2° C. In some embodiments, the pharmaceutical composition comprises at least about 90% G-CSF dimer, e.g., at least about 94% G-CSF dimer, as measured by nrCE-SDS after storage for up to about 3 months at 25±2° C. In some embodiments, the pharmaceutical composition comprises at least about 90% (e.g., at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) G-CSF dimer, e.g., at least about 94% G-CSF dimer, as measured by nrCE-SDS after storage for at least about 36 months at about 2° C. to about 8° C. In some embodiments, the pharmaceutical composition comprises at least about 90% G-CSF dimer, e.g., at least about 94% G-CSF dimer, as measured by nrCE-SDS after storage for up to about 36 months at about 2° C. to about 8° C.

[0121] In some embodiments, the pharmaceutical composition (e.g., a G-CSF-Fc dimer pharmaceutical composition, e.g., an efvemarenograstim alfa pharmaceutical composition) comprises at least about 90% (e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) G-CSF dimer, e.g., at least about 97% G-CSF dimer, as measured by SEC. In some embodiments, the pharmaceutical composition comprises at least about 85% (e.g., at least about 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) of the main peak based on the total area of all peaks as measured by SE-HPLC after storage at 25±2°C for at least about 1 month. In some embodiments, the pharmaceutical composition comprises at least about 85% (e.g., at least about any of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) of the main peak relative to the total area of all peaks when measured by SE-HPLC after at least about 2 months of storage at 25±2° C. In some embodiments, the pharmaceutical composition comprises at least about 85% of the main peak relative to the total area of all peaks when measured by SE-HPLC after at least about 3 months of storage at 25±2° C. In some embodiments, the pharmaceutical composition comprises at least about 85% (e.g., at least about any of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) of the main peak relative to the total area of all peaks when measured by SE-HPLC after at least about 36 months of storage at about 2° C. to about 8° C. In some embodiments, the pharmaceutical composition comprises at least about 85% of the main peak relative to the total area of all peaks when measured by SE-HPLC after up to about 36 months of storage at about 2° C. to about 8° C.

[0122] In some embodiments, the pharmaceutical composition (e.g., a G-CSF-Fc dimer pharmaceutical composition, e.g., an efvemarenograstim alfa pharmaceutical composition) comprises less than about 5% (e.g., less than about 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.5%, 1%, 0.5%, 0.1%, or less, e.g., less than about 2.5%) of the dimer relative to the total area of all peaks when measured by SE-HPLC after storage at 25±2° C. for at least about 1 month. In some embodiments, the pharmaceutical composition comprises less than about 5% (e.g., less than about 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.5%, 1%, 0.5%, 0.1%, or less, e.g., less than about 2.5%) of the dimer relative to the total area of all peaks when measured by SE-HPLC after storage at 25±2° C. for at least about 2 months. In some embodiments, the pharmaceutical composition comprises at least about less than 5% (e.g., less than about 2.5%) of dimer relative to the total area of all peaks when measured by SE-HPLC after storage for at least about 3 months at 25±2° C. In some embodiments, the pharmaceutical composition comprises at least about 5% (e.g., less than about 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.5%, 1%, 0.5%, 0.1%, or less, e.g., less than about 2.5%) of dimer relative to the total area of all peaks when measured by SE-HPLC after storage for at least about 36 months at about 2° C. to about 8° C. In some embodiments, the pharmaceutical composition comprises at least about less than 5% (e.g., less than about 2.5%) of dimer relative to the total area of all peaks when measured by SE-HPLC after storage for up to about 36 months at about 2° C. to about 8° C.

[0123] In some embodiments, the pharmaceutical composition (e.g., a G-CSF-Fc dimer pharmaceutical composition, e.g., an efvemarenograstim alfa pharmaceutical composition) contains about 2% or less (e.g., about 1.8%, 1.5%, 1%, 0.5%, 0.1%, or less) aggregates relative to the total area of all peaks when measured by SE-HPLC after storage at 25±2°C for at least about 1 month. In some embodiments, the pharmaceutical composition contains about 2% or less (e.g., about 1.8%, 1.5%, 1%, 0.5%, 0.1%, or less) aggregates relative to the total area of all peaks when measured by SE-HPLC after storage at 25±2°C for at least about 2 months. In some embodiments, the pharmaceutical composition contains about 2% or less aggregates relative to the total area of all peaks when measured by SE-HPLC after storage at 25±2°C for at least about 3 months. In some embodiments, the pharmaceutical composition comprises about 2% or less (e.g., about 1.8%, 1.5%, 1%, 0.5%, 0.1%, or less) aggregates relative to the total area of all peaks when measured by SE-HPLC after at least about 36 months of storage at about 2° C. to about 8° C. In some embodiments, the pharmaceutical composition comprises about 2% or less aggregates relative to the total area of all peaks when measured by SE-HPLC after up to about 36 months of storage at about 2° C. to about 8° C.

[0124] In some embodiments, the pharmaceutical composition (e.g., a liquid pharmaceutical composition) (e.g., a G-CSF-Fc dimer pharmaceutical composition, e.g., an efvemarenograstim alfa pharmaceutical composition) comprises about 27.2% to about 39.0% (e.g., about 30% to about 40%, e.g., about 32% to about 37%) of acidic region, about 36.5% to about 60.1% (e.g., about 45% to about 55%, e.g., about 47% to about 55%) of the main peak, and about 7.2% to about 30% (e.g., about 10% to about 20%, e.g., about 11% to 18%) of basic region, when measured by icIEF after storage at 25±2°C for at least about 1 month. In some embodiments, the pharmaceutical composition (e.g., liquid pharmaceutical composition) comprises about 27.2% to about 39.0% (e.g., about 30% to about 40%, e.g., about 32% to about 37%) acidic area, about 36.5% to about 60.1% (e.g., about 45% to about 55%, e.g., about 47% to about 55%) main peak, and about 7.2% to about 30% (e.g., about 10% to about 20%, e.g., about 11% to 18%) basic area when measured by icIEF after storage at 25±2° C. for at least about 2 months. In some embodiments, the pharmaceutical composition (e.g., liquid pharmaceutical composition) comprises about 27.2% to about 39.0% acidic area, about 36.5% to about 60.1% main peak, and about 7.2% to about 30% basic area when measured by icIEF after storage at 25±2° C. for at least about 3 months. In some embodiments, the pharmaceutical composition (e.g., liquid pharmaceutical composition) comprises about 27.2% to about 39.0% (e.g., about 30% to about 40%, e.g., about 32% to about 37%) acidic area, about 36.5% to about 60.1% (e.g., about 45% to about 55%, e.g., about 47% to about 55%) main peak, and about 7.2% to about 30% (e.g., about 10% to about 20%, e.g., about 11% to 18%) basic area, when measured by icIEF after storage at about 2°C to about 8°C for at least about 36 months. In some embodiments, the pharmaceutical composition (e.g., liquid pharmaceutical composition) comprises about 27.2% to about 39.0% acidic area, about 36.5% to about 60.1% main peak, and about 7.2% to about 30% basic area, when measured by icIEF after storage at about 2°C to about 8°C for up to about 36 months.

[0125] In some embodiments, the pharmaceutical composition (e.g., a G-CSF-Fc dimer pharmaceutical composition, e.g., an efvemarenograstim alfa pharmaceutical composition) retains at least about 70% (e.g., at least about 75%, 80%, 85%, 90%, 95%, 99%, 97%, 98%, 99%, or 100%) of its potency (e.g., G-CSF potency) after storage at 25±2°C for at least about 1 month. In some embodiments, the pharmaceutical composition retains at least about 70% (e.g., at least about 75%, 80%, 85%, 90%, 95%, 99%, or 100%) of its potency (e.g., G-CSF potency) after storage at 25±2°C for at least about 2 months. In some embodiments, the pharmaceutical composition retains at least about 70% potency (e.g., G-CSF potency) after at least about 3 months of storage at 25±2° C. In some embodiments, the pharmaceutical composition retains at least about 70% (e.g., at least about any of 75%, 80%, 85%, 90%, 95%, 99%, or 100%) potency (e.g., G-CSF potency) after at least about 36 months of storage at about 2° C. to about 8° C. In some embodiments, the pharmaceutical composition retains at least about 70% (e.g., G-CSF potency) after at least about 36 months of storage at about 2° C. to about 8° C.

[0126] In some embodiments, the change in concentration (e.g., G-CSF dimer concentration) of a pharmaceutical composition (e.g., a G-CSF-Fc dimer pharmaceutical composition such as an efvemarenograstim alfa pharmaceutical composition) is less than about 0.5 mg / mL (e.g., less than about any of 0.4 mg / mL, 0.3 mg / mL, 0.2 mg / mL, 0.1 mg / mL, or less) after at least about one month of storage at 25±2° C. In some embodiments, the change in concentration (e.g., G-CSF-Fc dimer concentration) of a pharmaceutical composition is less than about 0.5 mg / mL (e.g., less than about any of 0.4 mg / mL, 0.3 mg / mL, 0.2 mg / mL, 0.1 mg / mL, 0.05 mg / mL, or less) after at least about two months of storage at 25±2° C. In some embodiments, the change in concentration of the pharmaceutical composition (e.g., the concentration of G-CSF-Fc dimer) is less than about 0.5 mg / mL after at least about 3 months of storage at 25±2° C. In some embodiments, the change in concentration of the pharmaceutical composition (e.g., the concentration of G-CSF-Fc dimer) is less than about 0.5 mg / mL (e.g., less than about any of 0.4 mg / mL, 0.3 mg / mL, 0.2 mg / mL, 0.1 mg / mL, or less) after at least about 36 months of storage at about 2° C. to about 8° C. In some embodiments, the change in concentration of the pharmaceutical composition (e.g., the concentration of G-CSF-Fc dimer) is less than about 0.5 mg / mL after at least about 36 months of storage at about 2° C. to about 8° C.

[0127] In some embodiments, a commercial batch of any of the pharmaceutical compositions described herein (e.g., a G-CSF-Fc dimer pharmaceutical composition, e.g., an efvemarenograstim alfa pharmaceutical composition) is provided. As used herein, a "commercial batch" refers to a batch size of at least about 20 grams. A commercial batch is produced on a larger scale than a laboratory or bench-scale batch. The increased scale involves longer manufacturing times, including longer steps (such as evaporation steps) or longer wait times between steps. In some embodiments, the variation in the pharmaceutical composition between batches, e.g., the variation in any of the amount(s) of the pharmaceutical composition component(s), the pH of the pharmaceutical composition, the purity of the pharmaceutical composition, and one or more properties of the pharmaceutical composition (e.g., composition, stability, viscosity, osmolality, purity, and / or potency before and / or after storage), is about 5% or less (e.g., about 4%, 3%, 2%, 1%, 0.5%, 0.1%, or less).

[0128] Also provided are methods of producing any of the pharmaceutical compositions described herein (e.g., a G-CSF-Fc dimer pharmaceutical composition, e.g., an efvemarenograstim alfa pharmaceutical composition), comprising mixing the components of the pharmaceutical composition in the amounts described herein to produce the composition. In some embodiments, the pharmaceutical composition is a liquid pharmaceutical composition. In some embodiments, the pharmaceutical composition is a dry powder (e.g., a lyophilized pharmaceutical composition). In some embodiments, the pharmaceutical composition is a reconstituted pharmaceutical composition. In some embodiments, the method further comprises filling the pharmaceutical composition into a syringe (e.g., a sterile syringe). In some embodiments, the method further comprises purifying the G-CSF molecule (e.g., a G-CSF dimer) before mixing with other components of the pharmaceutical composition. In some embodiments, the method further comprises detecting (or verifying) one or more properties of the pharmaceutical composition, such as composition, stability, viscosity, osmolality, purity, and / or potency, before and / or after storage.

[0129] III. Methods for validating pharmaceutical compositions In another aspect, the present application provides a method for evaluating the suitability of any of the pharmaceutical compositions described herein (e.g., a G-CSF-Fc dimer pharmaceutical composition, e.g., an efvemarenograstim alfa pharmaceutical composition) for medical use in an individual (a human individual, e.g., a human with cancer and / or neutropenia). Such validated pharmaceutical compositions are also referred to herein as "suitable for medical use." In some embodiments, a method for evaluating the suitability of a pharmaceutical composition for medical use in an individual (a human individual, e.g., a human with cancer and / or neutropenia), the method comprising: (a) about 1 mg / mL to about 100 mg / mL of a G-CSF molecule (e.g., a -CSF-Fc dimer, e.g., efvemarenograstim alfa); (b) about 1 mM to about 50 mM of a buffering agent (e.g., sodium acetate); (c) about 0.1 mM to about 20 mM of a stabilizer (e.g., EDTA); and (d) about 1% (w / v) to about 15% (w / v) (e.g., about 1% (w / v) to about 10% (w / v)) of an isotonicity agent (e.g., erythrocyte monolayer, erythrocyte nuclei ... and (e) about 0.001% (w / v) to about 0.1% (w / v) of a surfactant (e.g., polysorbate 20), wherein the pharmaceutical composition has a pH of about 4.2 to about 6.2 (e.g., about 5.0 to about 5.4, e.g., about 5.2), and wherein the method comprises measuring a quality control parameter of the pharmaceutical composition and assessing the suitability of the pharmaceutical composition for medical use in an individual, wherein a quality control parameter measured within a quality control parameter threshold indicates the suitability of the pharmaceutical composition for medical use.In some embodiments, a method is provided for assessing the suitability of a pharmaceutical composition for medical use in an individual (a human individual, e.g., a human with cancer and / or neutropenia), the pharmaceutical composition comprising (or consisting essentially of, or consisting of) (a) about 20 mM G-CSF dimer (e.g., a G-CSF-Fc dimer, e.g., efvemarenograstim alfa); (b) about 10 mM sodium acetate; (c) about 1 mM EDTA; (d) about 5% (w / v) sorbitol; and (e) about 0.01% (w / v) polysorbate 20; and the pH of the pharmaceutical composition is about 5.2; the method comprises measuring a quality control parameter of the pharmaceutical composition; and assessing the suitability of the pharmaceutical composition for medical use in the individual, wherein a quality control parameter measured within a quality control parameter threshold indicates the suitability of the pharmaceutical composition for medical use. In some embodiments, the method is In the present specification, the G-CSF dimer comprises two monomeric subunits, each comprising (or consisting essentially of, or consisting of) the amino acid sequence of any of SEQ ID NOS: 6-10 (e.g., SEQ ID NO: 6). In some embodiments, the G-CSF dimer is efvemarenograstim alfa. The quality control parameter can be any one or combination of several parameters described herein for pharmaceutical compositions (e.g., composition, viscosity, stability, osmolality, purity, or potency), assessed, for example, by the corresponding methods described herein. One or more quality control parameters can be measured before and / or after storage. Any of the storage conditions described above in "II. Pharmaceutical Compositions" can be used for validation purposes herein. See also Example 8 and Table 14 for one or more quality control parameters, acceptance criteria (e.g., quality control parameter thresholds), and related methods.

[0130] In some embodiments, the quality control parameters include the appearance of the pharmaceutical composition (e.g., before and / or after storage). In some embodiments, the appearance of a clear, colorless to slightly yellowish solution, essentially free of visible particulates (e.g., about 10% or less, e.g., about 8%, 5%, 2%, 1%, or less) indicates the suitability of the pharmaceutical composition for medical use. In some embodiments, the pharmaceutical composition is placed in a colorimetric cuvette and visually inspected for visible particles against black and white backgrounds, respectively. In some embodiments, the pharmaceutical composition is compared to a colorimetric standard against a white background to assess color. In some embodiments, the quality control parameters are measured before storage. In some embodiments, the quality control parameters are measured after storage. In some embodiments, the storage conditions include storage at about 2°C to about 8°C for up to about 36 months. In some embodiments, the storage conditions include storage at 25±2°C for up to about 3 months.

[0131] In some embodiments, the quality control parameter includes the pH of the pharmaceutical composition (e.g., before and / or after storage). In some embodiments, a pH of the pharmaceutical composition of about 5.0 to about 5.4 (or 5.2±0.2, e.g., about 5.2) indicates the suitability of the pharmaceutical composition for medical use. In some embodiments, the pH is measured using a pH meter. In some embodiments, the quality control parameter is measured before storage. In some embodiments, the quality control parameter is measured after storage. In some embodiments, the storage conditions include storage at about 2°C to about 8°C for up to about 36 months. In some embodiments, the storage conditions include storage at 25±2°C for up to about 3 months.

[0132] In some embodiments, the quality control parameters include the size and / or quantity (e.g., particles per container) of particulates in the pharmaceutical composition (e.g., before and / or after storage). In some embodiments, the pharmaceutical composition is transferred to a vial, and the particles in the vial are inspected using a particle size measurement system (e.g., ACCUSize® 780 SIS). In some embodiments, suitability of the pharmaceutical composition for medical use is indicated when there are no more than 6,000 particles per vial with a diameter of 10 μm or greater and no more than 600 particles per vial with a diameter of 25 μm or greater. In some embodiments, the quality control parameters are measured before storage. In some embodiments, the quality control parameters are measured after storage. In some embodiments, the storage conditions include storage at about 2° C. to about 8° C. for up to about 36 months. In some embodiments, the storage conditions include storage at 25±2° C. for up to about 3 months.

[0133] In some embodiments, the quality control parameters include the composition of the pharmaceutical composition (e.g., before and / or after storage). In some embodiments, the method includes measuring one or more components of the pharmaceutical composition to assess the concentration and / or amount of the corresponding component. In some embodiments, the method includes measuring the pH of the pharmaceutical composition, for example, using a pH meter. Any suitable method for measuring a component(s) of the composition can be used herein, including, but not limited to, spectrophotometry (MS), rCE-SDS, nrCE-SDS, r-SDS-PAGE, nr-SDS-PAGE, SEC, HPLC (e.g., SE-HPLC, RP-HPLC), icIEF, or light scattering detection (LSD). In some embodiments, a variation in the concentration of each component and / or the pH of the pharmaceutical composition within about 5% (e.g., about any of 4%, 3%, 2%, 1%, 0.5%, 0.1%, or less) from the concentration and / or pH of each corresponding component described herein indicates the suitability of the pharmaceutical composition for medical use. In some embodiments, (a) a G-CSF dimer (e.g., a G-CSF-Fc dimer, e.g., efvemarenograstim alfa) that is about 1 mg / mL to about 100 mg / mL, (b) a buffering agent (e.g., sodium acetate) that is about 1 mM to about 50 mM, (c) a stabilizer (e.g., EDTA) that is about 0.1 mM to about 20 mM, (d) a tonicity agent (e.g., sorbitol) that is about 1% (w / v) to about 15% (w / v) (e.g., about 1% (w / v) to about 10% (w / v)), (e) a surfactant (e.g., polysorbate 20) that is about 0.001% (w / v) to about 0.1% (w / v), and (f) a pH of the pharmaceutical composition that is about 4.2 to about 6.2 (e.g., about 5.0 to about 5.4) indicate suitability of the pharmaceutical composition for medical use.In some embodiments, the composition contains: (a) a G-CSF dimer (e.g., a G-CSF-Fc dimer, e.g., efvemarenograstim alfa) that is about 18 mg / mL to about 22 mg / mL (or 20.0±2.0 mg / mL, e.g., about 20 mg / mL); (b) sodium acetate that is about 10 mM; (c) EDTA that is about 1 mM; (d) sorbitol that is about 5% (w / v); (e) polysorbate 20 that is about 0.01% (w / v); and (f) sorbitol that is about 5% (w / v); A pH of the pharmaceutical composition of about 5.4 (or 5.2±0.2, e.g., about 5.2) indicates suitability of the pharmaceutical composition for medical use. In some embodiments, the quality control parameter is measured before storage. In some embodiments, the quality control parameter is measured after storage. In some embodiments, the storage conditions include storage at about 2°C to about 8°C for up to about 36 months. In some embodiments, the storage conditions include storage at 25±2°C for up to about 3 months.

[0134] In some embodiments, the quality control parameter includes the potency of the pharmaceutical composition (e.g., before and / or after storage). In some embodiments, a potency (e.g., G-CSF potency) of about 70% to about 130% (e.g., about any of 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, or 130%), e.g., by a cell proliferation assay, indicates the suitability of the pharmaceutical composition for medical use. In some embodiments, the quality control parameter is measured before storage. In some embodiments, the quality control parameter is measured after storage. In some embodiments, the storage conditions include storage at about 2°C to about 8°C for up to about 36 months. In some embodiments, the storage conditions include storage at 25±2°C for up to about 3 months. Any suitable method for measuring G-CSF activity can be used herein, including, but not limited to, G-CSF receptor binding assays, reporter cell assays (e.g., using cells expressing G-CSF receptors to measure tyrosine phosphorylation of JAK kinases or transcription factors), cell proliferation and / or differentiation assays (e.g., using cells expressing G-CSF receptors, such as neutrophilic granulocytes), granulocyte colony formation assays, or in vivo assays (e.g., by measuring neutrophil counts). The activity of G-CSF can be determined by conventional activity tests as described in the art (see, for example, Draft Monographie "Filgrastim Concentrated Solution" Pharm Eur. Vol. 19, No. 1, January 2007, or Stute, N., et al. "Pharmacokinetics of subcutaneous recombinant human granulocyte colony-stimulating factor in children 1" (1992) Blood 79(11), pages 2849-2854).Measurement of G-CSF activity in vitro is described, for example, in Shirafuji, N. et al. (1989), "A new bioassay for human granulocyte colony-stimulating factor (hG-CSF) using murine myeloblastic NFS-60 cells as targets and estimation of its levels in serum from normal healthy persons and patients with infectious and hematological disorders," Exp. Hematol. (1989) 17, 116-119. Measurement of G-CSF activity in vivo is described, for example, in Tanaka, H. et al. (1991), "Pharmacokinetics of recombinant human granulocyte colony-stimulating factor conjugated to polyethylene glycol in rats," Cancer Research (1991) 51, 3710-3714. Further publications describing assays for measuring G-CSF activity include U.S. Patent No. 6,555,660 and Nohynek, GJ et al. 1997, Comparison of the potency of glycosylated and nonglycosylated recombinant human granulocyte colony-stimulating factors in neutropenic and normeutropenic CD rats, Cancer Chemother. Pharmacol. (1997) 39, 259-266. See also Example 8.

[0135] In some embodiments, the quality control parameters include the amount and / or percentage (%) of G-CSF dimer (e.g., before and / or after storage). In some embodiments, at least about 96% (e.g., at least about 97%, 98%, 99%, or 100%) of G-CSF dimer in the pharmaceutical composition as measured by SEC indicates suitability of the pharmaceutical composition for medical use. In some embodiments, a pharmaceutical composition comprising at least about 95.5% (e.g., at least about 96%, 97%, 98%, 99%, or 100%) of G-CSF dimer as measured by rCE-SDS indicates suitability of the pharmaceutical composition for medical use. In some embodiments, a pharmaceutical composition comprising at least about 94% (e.g., at least about 95%, 96%, 97%, 98%, 99%, or 100%) of G-CSF dimer as measured by nrCE-SDS indicates suitability of the pharmaceutical composition for medical use. In some embodiments, a pharmaceutical composition comprising about 1.5% or less (e.g., 1.4%, 1.3%, 1.2%, 1.1%, 1.0%, 0.8%, 0.5%, 0.3%, 0.1%, or less) HMWS as measured by rCE-SDS indicates the suitability of the pharmaceutical composition for medical use. In some embodiments, a pharmaceutical composition comprising about 1.5% or less (e.g., 1.4%, 1.3%, 1.2%, 1.1%, 1.0%, 0.8%, 0.5%, 0.3%, 0.1%, or less) HMWS as measured by nrCE-SDS indicates the suitability of the pharmaceutical composition for medical use. In some embodiments, a pharmaceutical composition comprising about 27.2% to about 39.0% (e.g., about 30% to about 40%, e.g., about 32% to about 37%) of an acidic region, about 36.5% to about 60.1% (e.g., about 45% to about 55%, e.g., about 47% to about 55%) of a main peak, and about 7.2% to about 30% (e.g., about 10% to about 20%, e.g., about 11% to 18%) of a basic region, as measured by icIEF, demonstrates suitability of the pharmaceutical composition for medical use.In some embodiments, a change in the concentration of the pharmaceutical composition (e.g., the concentration of the G-CSF-Fc dimer) by less than about 0.5 mg / mL (e.g., less than about any of 0.4 mg / mL, 0.3 mg / mL, 0.2 mg / mL, 0.1 mg / mL, 0.05 mg / mL, or less) indicates the suitability of the pharmaceutical composition for medical use. In some embodiments, a concentration of the pharmaceutical composition (e.g., the concentration of the G-CSF dimer) of 20.0±2.0 mg / mL (e.g., by UV spectrophotometer scanning) indicates the suitability of the pharmaceutical composition for medical use. In some embodiments, the quality control parameter is measured before storage. In some embodiments, the quality control parameter is measured after storage. In some embodiments, the storage conditions include storage at about 2° C. to about 8° C. for up to about 36 months. In some embodiments, the storage conditions include storage at 25±2° C. for up to about 3 months.

[0136] In some embodiments, the quality control parameter includes viscosity (e.g., before and / or after storage). In some embodiments, a viscosity range of the pharmaceutical composition at about 5°C to about 25°C in the range of about 1 to about 50 mPa·S, e.g., about 1 to about 45 mPa·S, about 1 to about 40 mPa·S, about 1 to about 35 mPa·S, about 1 to about 30 mPa·S, about 1 to about 25 mPa·S, or about 1 to about 20 mPa·S, about 1 to about 15 mPa·S, about 1 to about 10 mPa·S, about 1 to about 5 mPa·S, about 1 to about 4 mPa·S, about 1 to about 3 mPa·S, or about 1 to about 20 mPa·S, indicates the suitability of the pharmaceutical composition for medical use. For example, in some embodiments, a viscosity of about 1 to 2 mPa·S at about 25°C indicates the suitability of the pharmaceutical composition for medical use. In some embodiments, a pharmaceutical composition being isotonic indicates the suitability of the pharmaceutical composition for medical use. In some embodiments, the quality control parameter is measured before storage. In some embodiments, the quality control parameter is measured after storage. In some embodiments, the storage conditions include storage at about 2°C to about 8°C for up to about 36 months. In some embodiments, the storage conditions include storage at 25±2°C for up to about 3 months.

[0137] In some embodiments, the quality control parameter includes osmolality (e.g., before and / or after storage). In some embodiments, an osmolality of the pharmaceutical composition of about 100 to about 500 mOsm / kg, e.g., about 120 to about 480 mOsm / kg, about 140 to about 440 mOsm / kg, about 160 to about 420 mOsm / kg, about 180 to about 400 mOsm / kg, about 200 to about 380 mOsm / kg, about 220 to about 360 mOsm / kg, about 270 to about 350 mOsm / kg, or about 268 to about 360 mOsm / kg, indicates the suitability of the pharmaceutical composition for medical use. In some embodiments, an osmolality of the pharmaceutical composition of about 310 mOsm / kg indicates the suitability of the pharmaceutical composition for medical use. In some embodiments, the quality control parameter is measured before storage. In some embodiments, the quality control parameters are measured after storage. In some embodiments, the storage conditions include storage at about 2° C. to about 8° C. for up to about 36 months. In some embodiments, the storage conditions include storage at 25±2° C. for up to about 3 months.

[0138] In some embodiments, the quality control parameter includes the degree of protein aggregation (e.g., before and / or after storage). In some embodiments, a percent total protein aggregation of less than about 5%, e.g., less than about any of 4%, 3%, 2%, 1%, 0.5%, 0.2%, 0.1%, or less, indicates the suitability of the pharmaceutical composition for medical use. In some embodiments, a percent total protein aggregation of less than about 2% (e.g., less than any of 1.5%, 1%, 0.5%, 0.2%, 0.1%, or less), as measured by SEC, indicates the suitability of the pharmaceutical composition for medical use. In some embodiments, the quality control parameter is measured before storage. In some embodiments, the quality control parameter is measured after storage. In some embodiments, the storage conditions include storage at about 2°C to about 8°C for up to about 36 months. In some embodiments, the storage conditions include storage at 25±2°C for up to about 3 months.

[0139] In some embodiments, the quality control parameters include the percentage and / or amount (e.g., before and / or after storage) of HMWS. In some embodiments, a pharmaceutical composition comprising about 1.5% or less (e.g., about 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.8%, 0.5%, 0.1%, or less, e.g., about 1% or less) of HMWS as measured by rCE-SDS after at least about one month of storage at 25±2° C. indicates the suitability of the pharmaceutical composition for medical use. In some embodiments, a pharmaceutical composition comprising about 1.5% or less (e.g., about 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.8%, 0.5%, 0.1%, or less, e.g., 1% or less) of HMWS as measured by rCE-SDS after at least about two months of storage at 25±2° C. indicates the suitability of the pharmaceutical composition for medical use. In some embodiments, a pharmaceutical composition comprising about 1.5% or less (e.g., 1% or less) HMWS as measured by rCE-SDS after at least about 3 months of storage at 25±2° C. indicates the suitability of the pharmaceutical composition for medical use. In some embodiments, a pharmaceutical composition comprising about 1.5% or less (e.g., about 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.8%, 0.5%, 0.1%, or less) HMWS as measured by rCE-SDS after at least about 36 months of storage at about 2° C. to about 8° C. indicates the suitability of the pharmaceutical composition for medical use. In some embodiments, a pharmaceutical composition comprising about 1.5% or less HMWS as measured by rCE-SDS after up to about 36 months of storage at about 2° C. to about 8° C. indicates the suitability of the pharmaceutical composition for medical use. In some embodiments, a pharmaceutical composition comprising about 1.5% or less (e.g., about 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.8%, 0.5%, 0.1%, or less) HMWS as measured by rCE-SDS after storage at 25±2°C for at least about 1 month indicates the suitability of the pharmaceutical composition for medical use.In some embodiments, a pharmaceutical composition comprising about 1.5% or less (e.g., about 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.8%, 0.5%, 0.1%, or less) HMWS as measured by nrCE-SDS after at least about 2 months of storage at 25±2°C indicates suitability of the pharmaceutical composition for medical use. In some embodiments, a pharmaceutical composition comprising about 1.5% or less HMWS as measured by nrCE-SDS after up to about 3 months of storage at 25±2°C indicates suitability of the pharmaceutical composition for medical use. In some embodiments, a pharmaceutical composition comprising about 1.5% or less HMWS as measured by nrCE-SDS after at least about 36 months of storage at about 2°C to about 8°C indicates suitability of the pharmaceutical composition for medical use. In some embodiments, a pharmaceutical composition comprising about 1.5% or less HMWS as measured by nrCE-SDS after storage at about 2°C to about 8°C for up to about 36 months indicates suitability of the pharmaceutical composition for medical use.

[0140] In some embodiments, the quality control parameter includes the percentage and / or amount (e.g., before and / or after storage) of low molecular weight species (LMWS). In some embodiments, a pharmaceutical composition comprising about 4% or less (e.g., 3.5%, 3%, 2.5%, 2%, 1.5%, 1%, 0.8%, 0.5%, 0.1%, or less) LMWS as measured by rCE-SDS indicates the suitability of the pharmaceutical composition for medical use. In some embodiments, a pharmaceutical composition comprising about 6% or less (e.g., about 5.5%, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.5%, 1%, 0.8%, 0.5%, 0.1%, or less) LMWS as measured by nrCE-SDS indicates the suitability of the pharmaceutical composition for medical use. In some embodiments, the quality control parameter is measured before storage. In some embodiments, the quality control parameter is measured after storage. In some embodiments, the storage conditions include storage at about 2° C. to about 8° C. for up to about 36 months. In some embodiments, the storage conditions include storage at 25±2° C. for up to about 3 months.

[0141] In some embodiments, the quality control parameter includes the percentage and / or amount (e.g., before and / or after storage) of G-CSF dimer. In some embodiments, a pharmaceutical composition comprising at least about 90% (e.g., at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) G-CSF dimer indicates the suitability of the pharmaceutical composition for medical use. In some embodiments, the quality control parameter is measured before storage. In some embodiments, the quality control parameter is measured after storage. In some embodiments, the storage conditions include storage at about 2°C to about 8°C for up to about 36 months. In some embodiments, the storage conditions include storage at 25±2°C for up to about 3 months. In some embodiments, a pharmaceutical composition comprising at least about 95.5% (e.g., at least about any of 96%, 97%, 98%, 99%, or 100%) G-CSF dimer as measured by rCE-SDS after storage at 25±2° C. for at least about 1 month indicates the suitability of the pharmaceutical composition for medical use. In some embodiments, a pharmaceutical composition comprising at least about 95.5% (e.g., at least about any of 96%, 97%, 98%, 99%, or 100%) G-CSF dimer as measured by rCE-SDS after storage at 25±2° C. for at least about 2 months indicates the suitability of the pharmaceutical composition for medical use. In some embodiments, a pharmaceutical composition comprising at least about 95.5% G-CSF dimer as measured by rCE-SDS after storage at 25±2° C. for up to about 3 months indicates the suitability of the pharmaceutical composition for medical use. In some embodiments, a pharmaceutical composition comprising at least about 95.5% (e.g., at least about any of 96%, 97%, 98%, 99%, or 100%) G-CSF dimer as measured by rCE-SDS after storage at about 2° C. to about 8° C. for at least about 36 months indicates suitability of the pharmaceutical composition for medical use. In some embodiments, a pharmaceutical composition comprising at least about 95.5% G-CSF dimer as measured by rCE-SDS after storage at about 2° C. to about 8° C. for up to about 36 months indicates suitability of the pharmaceutical composition for medical use.In some embodiments, a pharmaceutical composition comprising at least about 94% (e.g., at least about any of 95%, 96%, 97%, 98%, 99%, or 100%) G-CSF dimer as measured by nrCE-SDS after storage at 25±2° C. for at least about 1 month indicates suitability of the pharmaceutical composition for medical use. In some embodiments, a pharmaceutical composition comprising at least about 94% (e.g., at least about any of 95%, 96%, 97%, 98%, 99%, or 100%) G-CSF dimer as measured by nrCE-SDS after storage at 25±2° C. for at least about 2 months indicates suitability of the pharmaceutical composition for medical use. In some embodiments, a pharmaceutical composition comprising at least about 94% G-CSF dimer as measured by nrCE-SDS after storage at 25±2° C. for up to about 3 months indicates suitability of the pharmaceutical composition for medical use. In some embodiments, a pharmaceutical composition comprising at least about 94% (e.g., at least about any of 95%, 96%, 97%, 98%, 99%, or 100%) G-CSF dimer as measured by nrCE-SDS after at least about 36 months of storage at about 2° C. to about 8° C. indicates suitability of the pharmaceutical composition for medical use. In some embodiments, a pharmaceutical composition comprising at least about 94% G-CSF dimer as measured by nrCE-SDS after up to about 36 months of storage at about 2° C. to about 8° C. indicates suitability of the pharmaceutical composition for medical use.

[0142] In some embodiments, the quality control parameter includes the percentage of G-CSF dimer (e.g., before and / or after storage) as measured by SEC. In some embodiments, a pharmaceutical composition comprising at least about 96% (e.g., at least about 97%, 98%, 99%, or 100%) G-CSF dimer as measured by SEC indicates the suitability of the pharmaceutical composition for medical use. In some embodiments, a pharmaceutical composition comprising at least about 96% (e.g., at least about 97%, 98%, 99%, or 100%) of the main peak relative to the total area of all peaks as measured by SE-HPLC indicates the suitability of the pharmaceutical composition for medical use. In some embodiments, the quality control parameter is measured before storage. In some embodiments, the quality control parameter is measured after storage. In some embodiments, the storage conditions include storage at about 2°C to about 8°C for up to about 36 months. In some embodiments, the storage conditions include storage at 25±2°C for up to about 3 months. In some embodiments, a pharmaceutical composition comprising at least about 96% (e.g., at least about any of 96%, 97%, 98%, 99%, or 100%) G-CSF dimer as measured by SE-HPLC after storage at 25±2° C. for at least about 1 month indicates the suitability of the pharmaceutical composition for medical use. In some embodiments, a pharmaceutical composition comprising at least about 96% (e.g., at least about any of 97%, 98%, 99%, or 100%) G-CSF dimer as measured by SE-HPLC after storage at 25±2° C. for at least about 2 months indicates the suitability of the pharmaceutical composition for medical use. In some embodiments, a pharmaceutical composition comprising at least about 96% G-CSF dimer as measured by SE-HPLC after storage at 25±2° C. for up to about 3 months indicates the suitability of the pharmaceutical composition for medical use. In some embodiments, a pharmaceutical composition comprising at least about 96% (e.g., at least about any of 97%, 98%, 99%, or 100%) G-CSF dimer as measured by SE-HPLC after at least about 36 months of storage at about 2°C to about 8°C indicates the suitability of the pharmaceutical composition for medical use.In some embodiments, a pharmaceutical composition comprising at least about 96% G-CSF dimer as measured by SE-HPLC after storage at about 2° C. to about 8° C. for up to about 36 months indicates the suitability of the pharmaceutical composition for medical use. In some embodiments, a pharmaceutical composition comprising at least about 85% (e.g., at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the main peak relative to the total area of all peaks as measured by SE-HPLC after storage at 25±2° C. for at least about 1 month indicates the suitability of the pharmaceutical composition for medical use. In some embodiments, a pharmaceutical composition comprising at least about 85% (e.g., at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the main peak relative to the total area of all peaks as measured by SE-HPLC after storage at 25±2° C. for at least about 2 months indicates the suitability of the pharmaceutical composition for medical use. In some embodiments, a pharmaceutical composition comprising at least about 85% of the total area of all peaks as measured by SE-HPLC after storage at 25±2° C. for up to about 3 months indicates the suitability of the pharmaceutical composition for medical use. In some embodiments, a pharmaceutical composition comprising at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the total area of all peaks as measured by SE-HPLC after storage at about 2° C. to about 8° C. for at least about 36 months indicates the suitability of the pharmaceutical composition for medical use. In some embodiments, a pharmaceutical composition comprising at least about 85% of the total area of all peaks as measured by SE-HPLC after storage at about 2° C. to about 8° C. for up to about 36 months indicates the suitability of the pharmaceutical composition for medical use.

[0143] In some embodiments, the quality control parameter includes the percentage of dimer (e.g., before and / or after storage) as measured by SEC. In some embodiments, a pharmaceutical composition comprising less than about 5% (e.g., less than about 4%, 3%, 2%, 1.5%, 1%, 0.5%, 0.1%, or less, e.g., less than about 2.5%) of dimer relative to the total area of all peaks as measured by SE-HPLC indicates the suitability of the pharmaceutical composition for medical use. In some embodiments, the quality control parameter is measured before storage. In some embodiments, the quality control parameter is measured after storage. In some embodiments, the storage conditions include storage at about 2°C to about 8°C for up to about 36 months. In some embodiments, the storage conditions include storage at 25±2°C for up to about 3 months. In some embodiments, a pharmaceutical composition comprising less than about 5% (e.g., less than about any of 4%, 3%, 2%, 1.5%, 1%, 0.5%, 0.1%, or less, e.g., less than about 2.5%) of dimer relative to the total area of all peaks when measured by SE-HPLC after storage at about 25±2° C. for at least about 1 month indicates suitability of the pharmaceutical composition for medical use. In some embodiments, a pharmaceutical composition comprising less than about 5% (e.g., less than about any of 4%, 3%, 2%, 1.5%, 1%, 0.5%, 0.1%, or less, e.g., less than about 2.5%) of dimer relative to the total area of all peaks when measured by SE-HPLC after storage at 25±2° C. for at least about 2 months indicates suitability of the pharmaceutical composition for medical use. In some embodiments, a pharmaceutical composition comprising less than about 5% (e.g., less than about 2.5%) of dimer relative to the total area of all peaks as measured by SE-HPLC after storage for up to about 3 months at 25±2° C. indicates suitability of the pharmaceutical composition for medical use. In some embodiments, a pharmaceutical composition comprising less than about 5% (e.g., less than about 4%, 3%, 2%, 1.5%, 1%, 0.5%, 0.1%, or less, e.g., less than about 2.5%) of dimer relative to the total area of all peaks as measured by SE-HPLC after storage for at least about 36 months at about 2° C. to about 8° C. indicates suitability of the pharmaceutical composition for medical use.In some embodiments, a pharmaceutical composition comprising less than about 5% (e.g., less than about 2.5%) of dimer relative to the total area of all peaks as measured by SE-HPLC after storage at about 2°C to about 8°C for up to about 36 months indicates the suitability of the pharmaceutical composition for medical use.

[0144] In some embodiments, the quality control parameters include peaks (e.g., before and / or after storage) measured by icIEF. In some embodiments, a pharmaceutical composition (e.g., a liquid pharmaceutical composition) comprising about 27.2% to about 39.0% (e.g., about 32% to about 37%) of acidic region, about 36.5% to about 60.1% (e.g., about 47% to about 55%) of main peak, and about 7.2% to about 30% (e.g., about 11% to about 18%) of basic region, as measured by icIEF after at least about 1 month of storage at 25±2° C., indicates the suitability of the pharmaceutical composition for medical use. In some embodiments, a pharmaceutical composition (e.g., a liquid pharmaceutical composition) comprising about 27.2% to about 39.0% (e.g., about 32% to about 37%) acidic area, about 36.5% to about 60.1% (e.g., about 47% to about 55%) main peak, and about 7.2% to about 30% (e.g., about 11% to about 18%) basic area as measured by icIEF after storage at 25±2° C. for at least about two months demonstrates suitability of the pharmaceutical composition for medical use. In some embodiments, a pharmaceutical composition (e.g., a liquid pharmaceutical composition) comprising about 27.2% to about 39.0% acidic area, about 36.5% to about 60.1% main peak, and about 7.2% to about 30% basic area as measured by icIEF after storage at 25±2° C. for up to about three months demonstrates suitability of the pharmaceutical composition for medical use. In some embodiments, a liquid pharmaceutical composition comprising about 27.2% to about 39.0% (e.g., about 32% to about 37%) acidic area, about 36.5% to about 60.1% (e.g., about 47% to about 55%) main peak, and about 7.2% to about 30% (e.g., about 11% to about 18%) basic area as measured by icIEF after storage at about 2° C. to about 8° C. for at least about 36 months demonstrates suitability of the pharmaceutical composition for medical use. In some embodiments, a liquid pharmaceutical composition comprising about 27.2% to about 39.0% acidic area, about 36.5% to about 60.1% main peak, and about 7.2% to about 30% basic area as measured by icIEF after storage at about 2° C. to about 8° C. for up to about 36 months demonstrates suitability of the pharmaceutical composition for medical use.

[0145] In some embodiments, the method comprises evaluating two or more of the quality control parameters set forth above, including, for example, at least any of 2, 3, 4, 5, 6, 7, 8, 9, 10, or more of the quality control parameters set forth above.

[0146] Also provided is a method of releasing a batch (e.g., a commercial batch) of a pharmaceutical composition described herein, comprising assessing the suitability of the pharmaceutical composition for medical use in an individual (e.g., a human individual) using a sample of the commercial batch according to any of the methods described herein (e.g., by assessing whether one or more quality control parameters described herein are within the quality control parameter threshold(s)), and releasing the commercial batch if the sampled pharmaceutical composition is suitable for medical use.

[0147] Also provided is a method of processing a sample of any of the pharmaceutical compositions described herein to verify that the sample is suitable for medical use in an individual (e.g., a human individual), comprising obtaining a sample from a commercial batch and using the sample from the commercial batch to assess the suitability of the sampled pharmaceutical composition for medical use in an individual (e.g., a human individual) according to any of the methods described herein, for example, by assessing whether one or more quality control parameters described herein are within the quality control parameter threshold(s).

[0148] Also provided is a commercial batch of any of the pharmaceutical compositions described herein, optionally evaluated, validated, released, or processed according to any of the methods described herein.

[0149] IV. G-CSF molecule Human G-CSF is a glycoprotein with 204 amino acids, including a 30-amino acid signal peptide. The mature G-CSF protein has a molecular weight of 18-20 kDa and consists of 174 amino acids without the signal peptide. It is secreted extracellularly. The human cells primarily responsible for this secretion are monocytes, fibroblasts, and endothelial cells. G-CSF has three major biological functions: 1) it acts on bone marrow precursors and stem cells to induce neutrophil differentiation, development, and maturation; 2) it activates mature neutrophils to participate in immune responses; and 3) it acts with other hematopoietic growth factors, such as stem cell factor, Flt-3 ligand, and GM-CSF, to mobilize hematopoietic stem cells.

[0150] The G-CSF receptor (G-CSFR) has been shown to be present in bone marrow hematopoietic stem cells (Sca+Lin-Th1low), progenitor cells (CD34+), committed granulocyte precursor cells, and mature neutrophils. G-CSFR is a specific receptor with high affinity for G-CSF and consists of 812 amino acids. Tamada et al. obtained the crystal structure of the G-CSF:G-CSFR complex and demonstrated a 2:2 stoichiometry of the G-CSF:G-CSFR complex by 2.8 Å diffraction analysis (PNAS, 2008, Vol. 103: 3135-3140). In other words, in each complex, each G-CSF binds to one receptor chain to form a G-CSF-receptor complex, and when two G-CSF-receptor complexes come into close proximity, this interaction results in the formation of a 2:2 dimer. In this context, the carboxyl terminus of the G-CSF receptor can activate downstream signaling molecules, JAK (Janus tyrosine kinase, mainly JAK2), which in turn activates STAT3 and switches on transcriptional genes important for neutrophil differentiation, proliferation, and activation.

[0151] In 2003, Schhabitz WR et al. reported that recombinant human G-CSF (rhG-CSF) was shown to have protective effects on neurons in an ischemic animal model (Storke, 2003, 34;745-751). Subsequently, in 2006, Schu et al. reported that rhG-CSF was shown to be clinically effective in treating patients with acute stroke who received daily rhG-CSF administration for five consecutive days (CMAJ, 2006, 174:927-933). The in vivo half-life of subcutaneously administered rat G-CSF is approximately 2 hours, whereas the half-life of subcutaneously administered human G-CSF is only 3.5 hours. Therefore, patients in need of treatment must receive daily drug administration or intravenous infusion, which affects their quality of life.

[0152] Pegfilgrastim, also known as Neulasta®, SD-01, and PEG-rmetHuG-CSF, is a G-CSF drug developed to reduce the severity and duration of severe neutropenia and its complications associated with the administration of myelosuppressive anticancer drugs or radiation therapy. Pegfilgrastim consists of recombinant methionyl human G-CSF (r-metHuG-CSF) covalently linked to monomethoxypolyethylene glycol (mPEG) via an amide bond. r-metHuG-CSF is identical to native human G-CSF (SEQ ID NO: 1) except for the N-terminal methionine required for expression in Escherichia coli. The attachment of mPEG to the N-terminal methionine of r-metHuG-CSF is believed to extend the serum half-life of r-metHuG-CSF (approximately 15-80 hours), thereby reducing the frequency of administration required to maintain therapeutic neutrophil counts.

[0153] ROLONTIS®, SPI-2012, HM10460A, and 17,65Efrapegrastim, also known as SG-CSF, is a long-acting G-CSF drug developed to reduce the severity and duration of severe neutropenia and the neutropenic complications associated with the use of myelosuppressive anticancer drugs or radiation therapy. Efrapegrastim consists of a recombinant human G-CSF analog (ef-G-CSF) and a recombinant fragment of the Fc region of human IgG4 linked by a bifunctional polyethylene glycol linker. ef-G-CSF differs from human G-CSF (SEQ ID NO: 1) by serine substitutions at positions 17 and 65. The Fc region of human IgG4 is believed to increase the serum half-life of ef-G-CSF to approximately 36.4 hours (range, approximately 16.1 to approximately 115 hours).

[0154] Both pegfilgrastim and efrapegrastim have the G-CSF molecule in monomeric form.

[0155] G-CSF-based drugs, such as filgrastim (Neupogen®) and its biosimilars, filgrastim-aafi (Nivestym®), filgrastim-sndz (Zarxio®), and filgrastim-ayow (Releuko®), are used to manage neutropenia in patients undergoing chemotherapy. However, these filgrastim and similar drugs have a short half-life of 3.5 hours, necessitating daily administration. Other G-CSF drugs, such as pegfilgrastim (e.g., Neulasta®) and efrapegrastim (e.g., ROLVEDON™), have long half-lives of 30–50 hours due to pegylation or fusion protein technology, allowing them to be administered once per chemotherapy cycle. Currently, the recommended dosing regimen for efrapegrastim (Rolontis®, HM10460A) and pegfilgrastim is administration the day after cytotoxic chemotherapy.

[0156] VG-CSF dimer In some embodiments, the G-CSF molecule is a G-CSF dimer. Any of the G-CSF dimers described herein can be used in the pharmaceutical compositions and methods of use described herein.

[0157] As used herein, the term "G-CSF dimer" refers to a protein comprising (or essentially consisting of) two units of a G-CSF molecule, e.g., two units of any of the G-CSF monomers described herein, or two units of a monomer subunit comprising any of the G-CSF molecules described herein. In a non-limiting example, a G-CSF dimer can comprise (or essentially consist of, or can comprise) two G-CSF monomers directly linked to each other or linked together via a linking moiety such as, for example, a peptide linker, a chemical bond, a covalent bond, or a polypeptide (e.g., a carrier protein, a dimerization domain). In another non-limiting example, a G-CSF dimer can comprise two monomer subunits, each comprising a G-CSF monomer connected to a carrier protein (e.g., albumin, or an Fc domain). Further examples of G-CSF dimers that can be used in the present invention are described in U.S. Pat. No. 8,557,546, U.S. Pat. No. 9,642,917, and U.S. Patent Application No. US20130165637, each of which is incorporated by reference in its entirety.

[0158] As used herein, the term "G-CSF monomer" refers to a single unit of the G-CSF protein or molecule. The terms "G-CSF," "G-CSF molecule," and "G-CSF protein" are used interchangeably herein.

[0159] The G-CSF monomer used in the G-CSF dimer can be derived from a G-CSF molecule of any organism, such as a human or a non-human animal, including, but not limited to, mammals such as primates (e.g., cynomolgus monkeys, rhesus monkeys), cows, pigs, horses, sheep, goats, rodents, cats, and dogs. The G-CSF monomer can be wild-type G-CSF or a mutant G-CSF, e.g., a mutant G-CSF capable of exhibiting most or all of the biological activity of wild-type G-CSF. In some embodiments, the G-CSF monomer is mature G-CSF. In some embodiments, the G-CSF monomer is a functional fragment of G-CSF capable of exhibiting most or all of the biological activity of full-length or mature G-CSF. In some embodiments, the G-CSF monomer is mouse G-CSF. In some embodiments, the G-CSF monomer is human G-CSF (hG-CSF). In some embodiments, the G-CSF monomer comprises (or consists of) the amino acid sequence of SEQ ID NO:1.

[0160] In some embodiments, the G-CSF dimer is a recombinant protein comprising two G-CSF (e.g., hG-CSF) molecules, for example, produced in a suitable host cell (e.g., a CHO cell). In some embodiments, the G-CSF dimer comprises (or consists essentially of) two G-CSF (e.g., hG-CSF) monomers, such as two G-CSF monomers connected to each other via a linker (e.g., a peptide linker). In some embodiments, the two G-CSF monomers forming the G-CSF dimer are the same (e.g., both comprise the sequence of SEQ ID NO: 1). In some embodiments, the two G-CSF monomers forming the G-CSF dimer are different.

[0161] In some embodiments, a G-CSF dimer comprises (or consists essentially of, or consists of) two G-CSF (e.g., hG-CSF) monomers and a carrier protein. In some embodiments, the carrier protein is albumin (e.g., human albumin). In some embodiments, the carrier protein is the Fc domain of an immunoglobulin (e.g., human IgG1, IgG2, IgG3, or IgG4). In some embodiments, the two G-CSF monomers forming the G-CSF dimer are the same (e.g., both comprise the sequence of SEQ ID NO: 1). In some embodiments, the two G-CSF monomers forming the G-CSF dimer are different.

[0162] In some embodiments, the G-CSF dimer comprises formula (I): M1-L-M2 (I) wherein M1 is a first monomer of G-CSF, M2 is a second monomer of G-CSF, and L is a linker disposed between the first and second monomers that connects them. In some embodiments, the linker L is selected from the group consisting of i) a short peptide comprising about 3 to about 50 amino acids, and ii) a polypeptide of formula (II): -ZYZ- (II) (wherein Y is a carrier protein (e.g., albumin, Fc domain), Z is null or a short peptide(s) comprising about 1 to about 30 amino acids, and "-" represents a chemical or covalent bond.) Any suitable linker or short peptide can be used herein that can provide flexibility between two G-CSF monomers or between a G-CSF monomer and a carrier protein and / or ensure binding of each G-CSF monomer to its receptor. In some embodiments, the linker L or the short peptide Z comprises the amino acid sequence of any of SEQ ID NOs: 12 to 31.

[0163] The carrier proteins described herein may be any protein suitable for linking two G-CSF monomers to form a G-CSF dimer, including, but not limited to, Fc fragments of immunoglobulins (e.g., human IgG1, IgG2, IgG3, IgG4) or albumin (e.g., human serum albumin). When a carrier protein is formed by linking two protein subunits (e.g., via a disulfide bond, peptide bond, or chemical bond), each protein subunit is referred to as a dimerization domain. In some embodiments, a carrier protein is formed by linking two dimerization domains (e.g., two Fc fragments of IgG) through one or more disulfide bonds. In some embodiments, the two dimerization domains forming the carrier protein are the same (e.g., two IgG2 Fc fragments). In some embodiments, the two dimerization domains forming the carrier protein are different. For example, in some embodiments, a carrier protein is formed by linking a first Fc fragment to a second, different Fc fragment through one or more disulfide bonds.

[0164] In some embodiments, a G-CSF dimer comprises (or consists essentially of, or consists of) two monomeric subunits, each of which comprises (or consists essentially of, or consists of) a G-CSF monomer (e.g., a human G-CSF monomer) and a dimerization domain. When the dimerization domain is an Fc fragment, such a G-CSF dimer is also referred to as a "G-CSF-Fc dimer."

[0165] In some embodiments, the G-CSF monomer comprises the amino acid sequence of SEQ ID NO: 1, or a variant thereof having at least about 90% sequence identity (e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the G-CSF monomer comprises the amino acid sequence of SEQ ID NO: 1.

[0166] In some embodiments, within each monomer subunit, the G-CSF monomer is directly connected to the dimerization domain.

[0167] In some embodiments, within each monomer subunit, the G-CSF monomer is connected to the dimerization domain via an optional linker. In some embodiments, the linker is about 6 to about 30 amino acids in length, e.g., about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids in length. In some embodiments, the linker is 16 amino acids in length. Any suitable linker that can provide flexibility between the G-CSF monomer and the dimerization domain can be used herein. In some embodiments, the linker comprises the amino acid sequence of any of SEQ ID NOs: 12-31. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 12.

[0168] The linker located between two G-CSF monomers or between one G-CSF monomer and a carrier protein or dimerization domain can be null (no linker), a peptide linker, or a non-peptide linker. In some embodiments, the first linker connecting the first G-CSF monomer and the carrier protein (or the first dimerization domain) is the same as the second linker connecting the second G-CSF monomer and the carrier protein (or the second dimerization domain). In some embodiments, the first linker connecting the first G-CSF monomer and the carrier protein (or the first dimerization domain) is different from the second linker connecting the second G-CSF monomer and the carrier protein (or the second dimerization domain). Generally, the linker does not affect or significantly affect the proper folding and conformation formed by the configuration of the two G-CSF monomers.

[0169] The linker may be a peptide linker of any length. In some embodiments, the peptide linker is about 1 amino acid (aa) to about 10 aa in length, about 2 aa to about 15 aa in length, about 5 aa to about 8 aa in length, about 1 aa to about 20 aa in length, about 21 aa to about 30 aa in length, about 1 aa to about 30 aa in length, about 10 aa to about 30 aa in length, about 3 aa to about 50 aa in length, or about 6 aa to about 30 aa in length. In some embodiments, the peptide linker is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length. In some embodiments, the peptide linker is about 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids in length. In some embodiments, the peptide linker is about any of 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids in length. For example, in some embodiments, the linker is between 3 and about 50 amino acids in length. In some embodiments, the linker is between about 6 and about 30 amino acids in length.

[0170] The peptide linker may have a naturally occurring or non-naturally occurring sequence. For example, a sequence derived from the hinge region of a heavy chain-only antibody can be used as a linker. See, for example, WO 1996 / 34103. In some embodiments, the peptide linker is a human IgG1, IgG2, IgG3, or IgG4 hinge. In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO: 13. In some embodiments, the linker is a flexible linker. Exemplary flexible linkers include, but are not limited to, glycine polymers (G) n (SEQ ID NO: 18), glycine-serine polymers (e.g., (GS) n (SEQ ID NO: 19), (GSGGS) n (SEQ ID NO: 20), or (GGGGS) n(SEQ ID NO: 22) (where n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers are relatively unstructured and can therefore function as neutral tethers between components. Glycine's accessibility in phi-psi space is significantly greater than alanine and is far less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11 173-142 (1992)). Exemplary flexible linkers include, but are not limited to, any of SEQ ID NOs: 12 and 14-31. In some embodiments, the linker comprises (or consists essentially of, or consists of) the amino acid sequence of SEQ ID NO: 12. In some embodiments, the linker (e.g., comprising SEQ ID NO: 12) further comprises the sequence of SEQ ID NO: 13. Those skilled in the art will recognize that the design of G-CSF dimers can include linkers that are wholly or partially flexible, such that the linker can include not only a flexible linker portion, but also one or more portions that confer a less flexible structure in providing the desired G-CSF dimer structure and function.

[0171] In some embodiments, the linker between the G-CSF monomer and the carrier protein (e.g., the dimerization domain) or between two G-CSF monomers is a stable linker (not cleaved by proteases, particularly MMPs).

[0172] Other linker considerations include their effect on the physical and pharmacokinetic properties of the resulting G-CSF dimer, such as solubility, lipophilicity, hydrophilicity, hydrophobicity, stability (some stability and planned degradability), rigidity, flexibility, immunogenicity, modulation of G-CSF / G-CSF receptor binding, and ability to be incorporated into micelles or liposomes.

[0173] In some embodiments, the dimerization domain (e.g., Fc fragment) in each monomer subunit contains at least two cysteines capable of forming intermolecular disulfide bonds. In some embodiments, there are about two to about four disulfide bonds between two dimerization domains (e.g., Fc fragments). In some embodiments, the dimerization domain comprises a leucine zipper. In some embodiments, the dimerization domain comprises at least a portion of an Fc fragment. In some embodiments, the Fc fragment comprises a CH2 and CH3 domain. In some embodiments, the dimerization domain is derived from an Fc fragment of any of IgA, IgD, IgE, IgG, and IgM, and their subtypes. In some embodiments, the Fc fragment is derived from an IgG1 Fc, IgG2 Fc, IgG4 Fc, or fragments or variants thereof. In some embodiments, the dimerization domain is derived from an Fc fragment of human IgG2. In some embodiments, the dimerization domain is derived from an Fc fragment of human IgG1. In some embodiments, the dimerization domain is derived from an Fc fragment of human IgG4. In some embodiments, the dimerization domain is a wild-type Fc fragment. In some embodiments, the dimerization domain comprises one or more mutations, such as mutations in the Fc fragment that reduce or eliminate effector function (e.g., decrease antibody-dependent cellular cytotoxicity (ADCC) or decrease binding to FcγR). In some embodiments, the Fc fragment comprises L234A and L235A ("LALA") substitutions (EU numbering). In some embodiments, the dimerization domain is an IgG2 Fc fragment comprising a P331S substitution according to EU numbering. In some embodiments, the IgG2 Fc fragment comprises the sequence of SEQ ID NO: 2 or 3. In some embodiments, the dimerization domain is an IgG1 Fc fragment comprising L234A, L235A, and P331S substitutions according to EU numbering. In some embodiments, the IgG1 Fc fragment comprises the sequence of SEQ ID NO: 4. In some embodiments, the dimerization domain is an IgG4 Fc fragment comprising S228P, L234A, and L235A substitutions according to EU numbering. In some embodiments, the IgG4 Fc fragment comprises the sequence of SEQ ID NO: 5.In some embodiments, the dimerization domain comprises a full-length Fc fragment. In some embodiments, the dimerization domain comprises an N-terminally truncated Fc fragment, e.g., a truncated Fc fragment with fewer N-terminal cysteines to reduce disulfide bond mispairing during dimerization. In some embodiments, the Fc fragment is N-terminally truncated, e.g., lacking the first 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids of a complete immunoglobulin Fc domain. In some embodiments, the dimerization domain is an IgG2 Fc fragment with the N-terminal "ERKCC" sequence (SEQ ID NO: 32) removed.

[0174] In some embodiments, each of the monomeric subunits comprises an Fc fragment of human IgG2 or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitutions. In some embodiments, each of the monomeric subunits comprises an Fc fragment of human IgG1 or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitutions. In some embodiments, each of the monomeric subunits comprises an Fc fragment of human IgG4 or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitutions. In some embodiments, each of the monomeric subunits comprises an Fc fragment having at least about 95% (e.g., at least about any of 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NOs: 2-5. In some embodiments, the Fc fragment comprises (or consists essentially of, or consists of) the sequence of SEQ ID NO: 2-5. In some embodiments, the Fc fragment comprises (or consists essentially of, or consists of) the amino acid sequence of SEQ ID NO: 2.

[0175] In some embodiments, the G-CSF dimer comprises two monomeric subunits, wherein i) each monomeric subunit comprises a G-CSF monomer comprising the amino acid sequence of SEQ ID NO: 1 and an Fc fragment comprising the amino acid sequence of SEQ ID NO: 2, ii) each monomeric subunit comprises a G-CSF monomer comprising the amino acid sequence of SEQ ID NO: 1 and an Fc fragment comprising the amino acid sequence of SEQ ID NO: 3, iii) each monomeric subunit comprises a G-CSF monomer comprising the amino acid sequence of SEQ ID NO: 1 and an Fc fragment comprising the amino acid sequence of SEQ ID NO: 4, or iv) each monomeric subunit comprises a G-CSF monomer comprising the amino acid sequence of SEQ ID NO: 1 and an Fc fragment comprising the amino acid sequence of SEQ ID NO: 5. In some embodiments, the G-CSF dimer comprises two monomeric subunits, wherein i) each monomeric subunit comprises a G-CSF monomer comprising the amino acid sequence of SEQ ID NO: 1 and an Fc fragment comprising the amino acid sequence of SEQ ID NO: 2. In some embodiments, within each monomer subunit, the G-CSF monomer is connected to the Fc fragment via a linker, such as a linker comprising the amino acid sequence of SEQ ID NO:12.

[0176] In some embodiments, the G-CSF monomer is located C-terminal to the dimerization domain in each monomer subunit. In some embodiments, the G-CSF monomer is located N-terminal to the dimerization domain in each monomer subunit.

[0177] In some embodiments, each monomeric subunit comprises (or consists essentially of, or consists of) the amino acid sequence of SEQ ID NO: 6, or a variant thereof having at least about 90% (e.g., at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid sequence of SEQ ID NO: 6. In some embodiments, each monomeric subunit comprises (or consists essentially of, or consists of) the amino acid sequence of SEQ ID NO: 7, or a variant thereof having at least about 90% (e.g., at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid sequence of SEQ ID NO: 7. In some embodiments, each monomeric subunit comprises (or consists essentially of, or consists of) the amino acid sequence of SEQ ID NO: 8, or a variant thereof having at least about 90% (e.g., at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid sequence of SEQ ID NO: 8. In some embodiments, each monomeric subunit comprises (or consists essentially of, or consists of) the amino acid sequence of SEQ ID NO: 9, or a variant thereof having at least about 90% (e.g., at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid sequence of SEQ ID NO: 9. In some embodiments, each monomeric subunit comprises (or consists essentially of, or consists of) the amino acid sequence of SEQ ID NO: 10, or a variant thereof having at least about 90% (e.g., at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid sequence of SEQ ID NO: 10.

[0178] In some embodiments, each monomeric subunit comprises (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 6-10. In some embodiments, each monomeric subunit comprises (or consists essentially of, or consists of) the amino acid sequence of SEQ ID NO: 6.

[0179] Thus, in some embodiments, the G-CSF dimer comprises two monomeric subunits, each comprising (or consisting essentially of, or consisting of) the amino acid sequence of SEQ ID NO: 6. This G-CSF dimer (or G-CSF-Fc dimer) is also referred to herein as "F-627" or "efvemarenograstim alfa." In some embodiments, each monomeric subunit of the G-CSF dimer is encoded by a nucleic acid comprising (or consisting essentially of, or consisting of) the sequence of SEQ ID NO: 11.

[0180] In some embodiments, an amino acid sequence that does not affect the biological activity of the G-CSF dimer can be added to the N- or C-terminus of one or both monomer subunits of the G-CSF dimer (e.g., the G-CSF monomer or the G-CSF-Fc monomer subunit). In some embodiments, such an added amino acid sequence is effective for enhancing expression (e.g., a signal peptide), purification (e.g., a 6xHis sequence, the cleavage site (Glu-Lys-Arg) of the Saccharomyces cerevisiae α-factor signal peptide), or biological activity of the G-CSF dimer.

[0181] In some embodiments, the G-CSF dimer is efvemarenograstim alfa. Efemarenograstim alfa is a long-acting fusion protein of human G-CSF and a human IgG2 Fc fragment, and exists as a dimer consisting of two G-CSF-Fc monomers covalently linked via a disulfide bond formed between the Fc fragments of the molecules. Each G-CSF-Fc monomer subunit comprises (or consists essentially of, or consists of) the amino acid sequence of SEQ ID NO: 6. Each hIgG2 Fc monomer subunit has a P297S substitution, as numbered from the N-terminus of the entire polypeptide chain of the G-CSF-Fc monomer subunit, which corresponds to a P331S substitution located in the CH2 domain according to EU numbering. The purpose of the P331S substitution is to reduce complement-dependent cytotoxicity (CDC) mediated by the Fc region of human IgG2. Efemarenograstim alfa is a glycosylated protein produced by Chinese hamster ovary cells using serum-free medium. Each G-CSF-Fc monomer subunit of efvemarenograstim alfa has one N-linked glycosylation site at N263, as numbered from the N-terminus of the entire polypeptide chain of the G-CSF-Fc monomer subunit, which corresponds to N297, located in the CH2 domain of the Fc region according to EU numbering. Each G-CSF-Fc monomer subunit also has one O-linked glycosylation site at T133 of the human G-CSF monomer. Without wishing to be bound by theory, it is believed that the IgG2 Fc fragment extends the serum half-life of human G-CSF.

[0182] By specifically binding to the G-CSF receptor, efevmarenograstim alfa stimulates the survival, proliferation, differentiation, and function of neutrophil precursors and mature neutrophils. Efevmarenograstim alfa was developed to reduce the incidence of infections manifesting as febrile neutropenia in patients with nonmyeloid malignancies receiving myelosuppressive anticancer drugs associated with clinically significant onset of febrile neutropenia (administration of efevmarenograstim alfa follows the myelosuppressive anticancer drug, e.g., 24 or 48 hours later).

[0183] a) Substitutions, insertions, deletions, and variants In some embodiments, G-CSF molecules (e.g., G-CSF dimers) having one or more amino acid mutations (e.g., substitutions) are provided. Conservative substitutions are shown in Table 1 under the heading "Preferred Substitutions." More substantial changes are shown in Table 1 under the heading "Exemplary Substitutions" and are further described below with reference to amino acid side chain classes. Amino acid substitutions can be introduced into G-CSF molecules (e.g., G-CSF dimers) (e.g., substitutions in one or more of the dimerization domains, such as the G-CSF monomer, linker, Fc fragment, etc.) and the products screened for a desired activity, e.g., retained / improved receptor binding, reduced immunogenicity, or improved ADCC or complement-dependent cytotoxicity (CDC). [Table 1]

[0184] Amino acids can be classified into groups based on common side chain properties: (1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain directionality: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.

[0185] Non-conservative substitutions involve exchanging a member of one of these classes for another class.

[0186] Modifications (eg, substitutions) can be made, for example, to improve G-CSF affinity.

[0187] In some embodiments, substitutions, insertions, or deletions may occur in a G-CSF molecule (e.g., a G-CSF dimer), as long as such modifications do not significantly reduce the ability of the G-CSF molecule to bind to a G-CSF receptor. For example, conservative modifications (e.g., conservative substitutions provided herein) that do not significantly reduce binding affinity may be made.

[0188] A useful method for identifying antibodies against G-CSF or variants thereof that can be targeted for mutagenesis is called "alanine scanning mutagenesis" and is described in Cunningham and Wells (1989) Science, 244:1081-1085. This method involves identifying a residue or group of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) and replacing it with a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the interaction of G-CSF with the receptor is affected. Further substitutions can be introduced at amino acid positions that demonstrate functional sensitivity to the initial substitution. Alternatively or additionally, a crystal structure of the receptor-ligand complex can be prepared to identify contact points between G-CSF and the receptor. These contact residues and neighboring residues can be targeted or removed as candidates for substitution. The variants can be screened to determine whether they have desired properties.

[0189] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Exemplary terminal insertions include fusing the N- or C-terminus of the G-CSF molecule to a polypeptide that increases the serum half-life of the G-CSF molecule.

[0190] b) Glycosylation variants In some embodiments, each G-CSF-Fc monomer subunit within a G-CSF dimer has one N-linked glycosylation site at N297, located in the CH2 domain according to EU numbering. In some embodiments, each G-CSF-Fc monomer subunit within a G-CSF dimer has one O-linked glycosylation site at T133 of the hG-CSF monomer relative to SEQ ID NO: 1.

[0191] In some embodiments, the G-CSF molecule (G-CSF dimer) is modified to increase or decrease the extent to which the protein is glycosylated. Glycosylation sites can be added or removed (e.g., to the Fc) conveniently by modifying the amino acid sequence such that one or more glycosylation sites are created or removed.

[0192] If the G-CSF dimer contains an Fc region, the carbohydrate attached thereto can be modified. Natural antibodies produced by mammalian cells typically contain branched, biantennary oligosaccharides, which are generally located at the C of the Fc region. H The Fc region is N-linked to Asn297 in the Fc domain. See, e.g., Wright et al., TIBTECH 15:26-32 (1997). The oligosaccharides can contain various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose linked to GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, oligosaccharide modifications in the Fc region can be made to generate Fc variants with improved specific properties.

[0193] In some embodiments, a G-CSF-Fc dimer has a carbohydrate structure lacking fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such a G-CSF-Fc dimer can be about 1% to about 80%, about 1% to about 65%, about 5% to about 65%, or about 20% to about 40%. For example, as described in WO 2008 / 077546, the amount of fucose is determined by calculating the average amount of fucose at N297 in the glycan relative to the sum of all glycan structures (e.g., complex, hybrid, and high-mannose structures) attached to N297 as measured by MALDI-TOF mass spectrometry. N297 refers to an asparagine residue located approximately at position 297 (in EU numbering of Fc region residues) within the Fc region; however, due to minor sequence variations in the Fc domain, N297 may also be located approximately ±3 amino acids upstream or downstream from position 297, i.e., between positions 294 and 300. Such fucosylated variants may have improved ADCC function. See, for example, U.S. Patent Application Publication Nos. US2003 / 0157108 (Presta, L.); US2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd.). Examples of publications relating to "defucosylated" or "fucose-deficient" proteins include US2003 / 0157108, WO2000 / 61739, WO2001 / 29246, US2003 / 0115614, US2002 / 0164328, US2004 / 0093621, US2004 / 0132140, US2004 / 0110704, US2004 / 0110282, US2004 / 0109865, WO2003 / 085119, WO2003 / 084570, WO2005 / 035586, WO2005 / 035778, WO2005 / 053742, WO2002 / 031140, Okazaki et al. J. Mol. Biol. 336:1239-1249(2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87:614(2004).Examples of cell lines capable of producing defucosylated proteins include Lec13 CHO cells, which are deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application No. US2003 / 0157108A1, Presta, L; and WO2004 / 056312A1, Adams et al., especially Example 11), and knockout cell lines, such as CHO cells in which the alpha-1,6-fucosyltransferase gene FUT8 has been knocked out (e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003 / 085107).

[0194] In some embodiments, the G-CSF molecule (e.g., a G-CSF dimer, e.g., a G-CSF-Fc dimer) has bisected oligosaccharides, e.g., a biantennary oligosaccharide attached to the Fc region of an antibody is bisected by GlcNAc. Such variants may have reduced fucosylation and / or improved ADCC function.

[0195] c) Fc region variants In some embodiments, the Fc fragment of the G-CSF-Fc dimer has reduced effector function compared to the corresponding wild-type Fc domain (e.g., at least about any of a 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, or 95% reduction as measured by the level of antibody-dependent cellular cytotoxicity (ADCC)).

[0196] In some embodiments, one or more amino acid modifications can be introduced into the Fc region of a G-CSF-Fc dimer to generate an Fc region variant, which can comprise a human Fc region sequence containing an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0197] In some embodiments, the Fc fragment retains some, but not all, effector functions, making it a desirable candidate for applications in which the in vivo half-life of the G-CSF-Fc dimer is important but certain effector functions (e.g., complement and ADCC) are unnecessary or deleterious. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduced / depleted CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to demonstrate that the G-CSF-Fc dimer lacks FcγR binding (and thus likely lacks ADCC activity) but retains FcRn binding ability. Natural killer (NK) cells, the primary cells mediating ADCC, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 2 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362 (e.g., Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); 5,821,337 (See Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods can be employed (e.g., the ACTI™ Non-Radioactive Cytotoxicity Assay for Flow Cytometry (CellTechnology, Inc. Mountain View, CA); and CytoTox 96™). 商標See, e.g., the ) non-radioactive cytotoxicity assay (Promega, Madison, WI). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and NK cells. Alternatively, or additionally, ADCC activity of the molecule of interest can be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays can also be performed to confirm that the G-CSF-Fc dimer is unable to bind C1q and therefore lacks CDC activity. See, e.g., the C1q and C3c binding ELISAs in WO2006 / 029879 and WO2005 / 100402. To assess complement activation, a CDC assay can be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). Determination of FcRn binding and in vivo clearance / half-life can also be performed using methods well known in the art (see, e.g., Petkova et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0198] Fc domains with reduced effector function include those with substitutions of one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc mutant with substitutions of residues 265 and 297 to alanine (U.S. Patent No. 7,332,581). In some embodiments, the Fc fragment comprises an N297A mutation. In some embodiments, the Fc fragment comprises an N297G mutation.

[0199] Certain Fc variants with improved or diminished binding to FcRs have been described (see, e.g., U.S. Patent No. 6,737,056, WO 2004 / 056312, and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001)).

[0200] In some embodiments, modifications are made to the Fc region that result in altered (either improved or decreased) C1q binding and / or CDC, e.g., as described in U.S. Pat. No. 6,194,551, WO 99 / 51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000).

[0201] In some embodiments, the G-CSF-Fc dimer comprises a variant Fc region containing one or more amino acid substitutions that alter half-life and / or alter binding to the fetal Fc receptor (FcRn). Fc domains with extended half-life and improved binding to FcRn, responsible for transport of maternal IgG to the fetus, are described in US2005 / 0014934A1 (Hinton et al.) (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)). These Fc domains with one or more substitutions, such as a substitution at Fc region residue 434, have altered binding of the Fc region to FcRn (U.S. Patent No. 7,371,826).

[0202] For other examples of Fc region variants, see also Duncan & Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94 / 29351.

[0203] In some embodiments, it may be desirable to generate cysteine-engineered G-CSF dimers in which one or more residues of the G-CSF dimer are replaced with cysteine residues. In specific embodiments, the replaced residues are located at accessible sites of the G-CSF dimer. By replacing these residues with cysteine, reactive thiol groups are placed at accessible sites of the G-CSF dimer, which can be used to link the G-CSF dimer to other moieties. In some embodiments, any one or more of the following residues can be replaced with cysteine: A118 (EU numbering) in the hinge and S400 (EU numbering) in the Fc region. Cysteine-engineered G-CSF dimers can be generated, for example, as described in U.S. Patent No. 7,521,541. Engineered G-CSF dimer variants can provide a variety of inter- and intramolecular linkages.

[0204] In some embodiments, the C-terminal lysine (K447) of the Fc region can be cleaved by endogenous carboxypeptidase digestion in mammalian cell culture. The absence of the C-terminal lysine does not affect the structure or stability of the Fc region. See Harris et al., Journal of Chromatography A, 705(1995)129-134. In some embodiments, the G-CSF dimer comprises an Fc region from which the C-terminal lysine residue has been cleaved. In some embodiments, a pharmaceutical composition is provided comprising a mixture of G-CSF dimers comprising an Fc region, wherein at least some of the G-CSF dimers comprise an Fc region from which the C-terminal lysine residue has been cleaved.

[0205] VI. Treatment method Also provided herein are methods for treating or preventing a disease or condition, or modulating an immune response, in an individual (e.g., a human) in need thereof. The methods comprise administering to the individual an effective amount of any of the G-CSF dimer pharmaceutical compositions described herein (e.g., a G-CSF-Fc dimer pharmaceutical composition, such as an efvemarenograstim alfa pharmaceutical composition). In some embodiments, the disease or condition is selected from the group consisting of neutropenia, stroke, spinal cord injury, neurological disorders associated with blood-brain barrier damage, Parkinson's disease, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, spinal muscular atrophy, primary lateral sclerosis, spinocerebellar ataxia, and mobilization of hematopoietic stem cells into peripheral blood in allogeneic blood stem cell transplantation. In some embodiments, the disease or condition is characterized by impaired white blood cell production. In some embodiments, the disease or condition is neutropenia, such as chemotherapy-induced neutropenia or radiation therapy-induced neutropenia. In some embodiments, the disease or condition is mobilization of hematopoietic stem cells into peripheral blood in allogeneic blood stem cell transplantation. In some embodiments, the pharmaceutical composition is administered at a dose of about 0.01 mg / kg to about 1 mg / kg. In some embodiments, the pharmaceutical composition is administered subcutaneously. In some embodiments, the pharmaceutical composition is administered once every three weeks.

[0206] In some embodiments, a method for treating or preventing a disease or condition (e.g., a condition characterized by impaired white blood cell production) in an individual (a human, e.g., a human with cancer) includes administering to the individual an effective amount of a pharmaceutical composition, the pharmaceutical composition comprising: (a) about 1 mg / mL to about 100 mg / mL (e.g., about 5 mg / mL to about 50 mg / mL, or about 20 mg / mL, etc.) of a G-CSF dimer (e.g., a G-CSF-Fc dimer, e.g., efvemarenograstim alfa); and (b) about 1 mM to about 50 mM (e.g., about 1 mM to about 30 mM (c) about 0.1 mM to about 20 mM (e.g., about 0.1 mM to about 10 mM, or about 1 mM) of a buffering agent (e.g., sodium acetate); (d) about 1% (w / v) to about 15% (w / v) (e.g., about 1% (w / v) to about 10% (w / v), about 5% (w / v), or about 9% (w / v)) of an isotonicity agent (e.g., sorbitol or sucrose); and (e) about 0.001% (w / v) to about 0.1% (w / v) (e.g., about 0.005% (w / v) to about 0.05% (w / v), or about 0.01 % (w / v) of a surfactant (e.g., polysorbate 20 or polysorbate 80), and the pH of the pharmaceutical composition is about 4.2 to about 6.2 (e.g., about 4.8 to about 5.8, about 5.0 to about 5.4, or about 5.2). In some embodiments, a method is provided for treating or preventing a disease or condition (e.g., a condition characterized by impaired white blood cell production) in an individual (e.g., a human, such as a human with cancer), comprising administering to the individual an effective amount of the pharmaceutical composition. and (e) about 0.01% (w / v) polysorbate 20; and wherein the pharmaceutical composition comprises (or consists essentially of, or consists of) (a) about 18 mg / mL to about 22 mg / mL (e.g., about 20 mg / mL) of a G-CSF dimer (e.g., a G-CSF-Fc dimer, e.g., efvemarenograstim alfa), (b) about 10 mM sodium acetate, (c) about 1 mM EDTA, (d) about 5% (w / v) sorbitol, and (e) about 0.01% (w / v) polysorbate 20; and wherein the pH of the pharmaceutical composition is about 5.0 to about 5.4 (e.g., about 5.2).In some embodiments, a method for treating or preventing a disease or condition (e.g., a condition characterized by impaired white blood cell production) in an individual (e.g., a human, such as a human with cancer) comprises administering to the individual an effective amount of a pharmaceutical composition, the pharmaceutical composition comprising: (a) about 20 mg / mL G-CSF dimer (e.g., G-CSF-Fc dimer, e.g., efvemarenograstim alfa); (b) about 10 mM sodium acetate; (c) about 1 mM EDTA; (d) about 5% (w / v) sorbitol; and (e) about 0.01% (w / v) polysorbate 80 (PBMC). and resorbate 20, and the pH of the pharmaceutical composition is about 5.2. In some embodiments, the G-CSF dimer comprises two monomeric subunits, each comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the G-CSF dimer comprises two monomeric subunits, each comprising a G-CSF monomer (SEQ ID NO: 1), an Fc fragment (e.g., any of SEQ ID NOs: 2-5), and a nucleotide sequence connecting the G-CSF monomer and the Fc fragment. , and an optional linker (SEQ ID NO: 12). In some embodiments, the G-CSF dimer comprises two monomeric subunits, each comprising (or consisting essentially of, or consisting of) the amino acid sequence of any of SEQ ID NOs: 6-10 (e.g., SEQ ID NO: 6). In some embodiments, the G-CSF dimer is efvemarenograstim alfa. In some embodiments, the pharmaceutical composition is contained in a syringe (e.g., sterile and / or single-use). In some embodiments, the volume of the pharmaceutical composition in the syringe is about 1 mL. In some embodiments, the pharmaceutical composition is administered subcutaneously. In some embodiments, the disease or condition is neutropenia, such as chemotherapy-induced neutropenia or radiation therapy-induced neutropenia. In some embodiments, the method comprises administering an effective amount of the pharmaceutical composition to the individual after (e.g., 24 hours or 48 hours after) administration of a chemotherapeutic agent to the individual. In some embodiments, the chemotherapeutic agent is a myelosuppressive chemotherapeutic agent. In some embodiments, the pharmaceutical composition is administered once every three weeks.In some embodiments, the individual is administered at least four cycles (e.g., about 21 days per cycle) of chemotherapy, and the pharmaceutical composition is administered after (e.g., 24 hours or 48 hours after) administration of the chemotherapy in each of the at least four cycles. In some embodiments, the pharmaceutical composition is administered at about 5 mg to about 25 mg (e.g., about 20 mg) of G-CSF dimer per administration (e.g., once per chemotherapy cycle).

[0207] The methods described herein can be used to treat and / or prevent one or more chemotherapy-induced or chemotherapy-associated conditions, including, but not limited to, conditions characterized by a decrease in absolute neutrophil count (ANC) (e.g., a decrease of at least about any of 5%, 10%, 20%, 50%, 60%, 70%, 80%, 90%, or more), a decrease in granulocyte count, a decrease in stem cell production, a decrease in hematopoiesis, a decrease in hematopoietic progenitor cell (HPC) count, and impaired white blood cell production.

[0208] In some embodiments, the disease or condition is characterized by impaired white blood cell production. In some embodiments, the condition characterized by impaired white blood cell production is neutropenia. In some embodiments, the condition characterized by impaired white blood cell production is chemotherapy-induced neutropenia or radiation therapy-induced neutropenia. In some embodiments, the neutropenia is drug-induced neutropenia. In some embodiments, the neutropenia is anti-cancer drug-induced neutropenia. In some embodiments, the anti-cancer drug is myelosuppressive. In some embodiments, the neutropenia is induced by a myelosuppressive anti-cancer drug.

[0209] In some embodiments, methods are provided for treating or preventing a condition characterized by impaired white blood cell production (e.g., chemotherapy-induced neutropenia or infection) in an individual (e.g., a human, e.g., a human with cancer), comprising administering to the individual an effective amount of any of the pharmaceutical compositions described herein (e.g., a G-CSF-Fc dimer pharmaceutical composition, such as efvemarenograstim alfa) following (e.g., at least 24 hours after) administration of a chemotherapeutic agent to the individual. In some embodiments, a method of treating or preventing a condition characterized by impaired white blood cell production (e.g., chemotherapy-induced neutropenia or an infection) in an individual (e.g., a human, e.g., a human with cancer) comprises administering to the individual an effective amount of a pharmaceutical composition described herein after (e.g., at least 24 hours after) administration of a chemotherapy agent to the individual, wherein the pharmaceutical composition comprises (or consists essentially of, or consists of) (a) about 20 mg / mL G-CSF dimer (e.g., G-CSF-Fc dimer, e.g., efvemarenograstim alfa); (b) about 10 mM sodium acetate; (c) about 1 mM EDTA; (d) about 5% (w / v) sorbitol; and (e) about 0.01% (w / v) polysorbate 20. ), and the pH of the pharmaceutical composition is about 5.2. In some embodiments, methods are provided for increasing (e.g., by at least about any of 10%, 20%, 50%, 70%, 90%, 1-fold, 2-fold, 5-fold, 20-fold, or more) one or more of i) ANC, ii) granulocyte count (e.g., in a subject eligible for bone marrow transplant), iii) stem cell production, iv) hematopoiesis, and v) HPC count in an individual in need thereof (e.g., a human, e.g., an individual with cancer), comprising administering to the individual an effective amount of any of the pharmaceutical compositions described herein (e.g., a G-CSF-Fc dimer pharmaceutical composition, such as an efvemarenograstim alfa pharmaceutical composition) after (e.g., at least 24 hours after) administration of a chemotherapeutic agent to the individual.In some embodiments, provided are methods of reducing (e.g., by at least about any of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more) the duration of chemotherapy-induced neutropenia (e.g., Grade 4) in an individual in need thereof (e.g., a human, e.g., an individual having cancer), comprising administering to the individual an effective amount of any of the pharmaceutical compositions described herein (e.g., a G-CSF-Fc dimer pharmaceutical composition, such as an efvemarenograstim alfa pharmaceutical composition) following (e.g., at least 24 hours after) administration of a chemotherapy agent to the individual. In some embodiments, methods are provided for reducing or preventing (e.g., reducing by at least about any of about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more) the occurrence of chemotherapy-induced neutropenia (e.g., Grade 4) in an individual in need thereof (e.g., a human, e.g., an individual with cancer), comprising administering to the individual an effective amount of any of the pharmaceutical compositions described herein (e.g., a G-CSF-Fc dimer pharmaceutical composition, such as an efvemarenograstim alfa pharmaceutical composition) after (e.g., at least 24 hours after) administration of a chemotherapy agent to the individual. In some embodiments, the method comprises ... a G-CSF-Fc dimer pharmaceutical composition, such as an efvemarenograstim alfa pharmaceutical composition, after (e.g., at least 24 hours after) administration of a chemotherapy agent to the individual. In some embodiments, the method comprises administering to the individual a G-CSF-Fc dimer pharmaceutical composition, such as an efvemarenograstim alfa pharmaceutical composition, after (e.g., at least 24 hours after) administration of a chemotherapy agent to the individual. 9 / L to less than about 36 hours (e.g., less than about any of 34, 32, 30, 28, 26, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.5, 0.2, 0.1 hours or less). In some embodiments, the method reduces the duration of an ANC of less than about 0.5 x 10 9 / L (e.g., at least about 0.4 × 10 9 / L, 0.2 × 10 9 / L, 0.1 × 10 9 / L, 1.0 × 10 7 / L, or less, whichever is less. In some embodiments, administration of the G-CSF dimer pharmaceutical composition prevents the ANC from reaching about 0.5 x 10 9 / L or less within about 10 days (e.g., within about 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.2, 0.1 days, or less) of the first occurrence of 9 / L or greater. In some embodiments, administration of the G-CSF dimer increases the ANC by about 0.5 x 10 9 / L or less within about 10 days (e.g., within about 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.2, 0.1 days, or less) of the first occurrence of 9 / L or more. In some embodiments, provided are methods for reducing or preventing (e.g., reducing by at least about any of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more) the occurrence of febrile neutropenia (FN) in an individual in need thereof (e.g., a human, e.g., an individual with cancer), comprising administering to the individual an effective amount of any of the pharmaceutical compositions described herein (e.g., a G-CSF-Fc dimer pharmaceutical composition, such as an efvemarenograstim alfa pharmaceutical composition) following (e.g., at least 24 hours after) administration of a chemotherapeutic agent to the individual. In some embodiments, provided are methods of activating one or more of i) neutrophil progenitor cells, ii) bone marrow stem cells, and iii) mature neutrophils in an individual in need thereof (e.g., a human, e.g., an individual having cancer), the method comprising administering to the individual an effective amount of any of the pharmaceutical compositions described herein (e.g., a G-CSF-Fc dimer pharmaceutical composition, such as an efvemarenograstim alfa pharmaceutical composition) following (e.g., at least 24 hours after) administration of a chemotherapeutic agent to the individual. In some embodiments, the pharmaceutical composition comprises (or consists essentially of, or consists of) (a) about 20 mM G-CSF dimer (e.g., G-CSF-Fc dimer, e.g., efvemarenograstim alfa), (b) about 10 mM sodium acetate, (c) about 1 mM EDTA, (d) about 5% (w / v) sorbitol, and (e) about 0.01% (w / v) polysorbate 20, and the pH of the pharmaceutical composition is about 5.2. In some embodiments, the G-CSF The dimer comprises two monomeric subunits, each comprising (or consisting essentially of, or consisting of) the amino acid sequence of any of SEQ ID NOS: 6-10 (e.g., SEQ ID NO: 6). In some embodiments, the G-CSF dimer is efvemarenograstim alfa. In some embodiments, the method comprises administering to the individual an effective amount of the pharmaceutical composition at least 24 hours after administration of a chemotherapeutic agent to the individual. In some embodiments, the chemotherapeutic agent is a myelosuppressive chemotherapeutic agent.In some embodiments, the myelosuppressive chemotherapeutic agent is selected from the group consisting of epirubicin, docetaxel, cyclophosphamide, doxorubicin, etoposide, cisplatin, paclitaxel, topotecan, vincristine, methylprednisolone, cytarabine, and combinations thereof. In some embodiments, the individual is administered two or more chemotherapeutic agents including: i) epirubicin and cyclophosphamide, ii) docetaxel and cyclophosphamide, iii) doxorubicin and cyclophosphamide, iv) docetaxel and doxorubicin, or v) docetaxel, doxorubicin, and cyclophosphamide. In some embodiments, the individual is administered a chemotherapeutic agent for treating cancer. In some embodiments, the cancer is selected from the group consisting of breast cancer, non-small cell lung cancer, small cell lung cancer, ovarian cancer, sarcoma, urothelial carcinoma, germ cell tumors, and non-Hodgkin's lymphoma. In some embodiments, the individual has been administered at least four cycles of chemotherapy, and the pharmaceutical composition is administered after (e.g., at least 24 hours after) administration of the chemotherapy in each of the at least four cycles. In some embodiments, the individual has breast cancer, and (i) the individual has been administered at least four cycles of docetaxel and cyclophosphamide, and docetaxel 75 mg / m. 2 and cyclophosphamide 600 mg / m 2 is administered on day 1 of each cycle and the pharmaceutical composition is administered on day 2 of each cycle (e.g., at least 24 hours after administration of docetaxel and cyclophosphamide), or (ii) the individual is administered at least four cycles of epirubicin and cyclophosphamide and epirubicin 100 mg / m 2 and cyclophosphamide 600 mg / m 2 is administered on day 1 of each cycle and the pharmaceutical composition is administered on day 3 of each cycle (e.g., at least 48 hours after administration of epirubicin and cyclophosphamide), or (iii) the individual is administered at least four cycles of docetaxel and doxorubicin and the pharmaceutical composition is administered on day 3 of each cycle (e.g., at least 48 hours after administration of epirubicin and cyclophosphamide), or 2 and doxorubicin 60 mg / m 2is administered on day 1 of each cycle, and the pharmaceutical composition is administered on day 2 of each cycle (e.g., at least 24 hours after administration of docetaxel and doxorubicin). In some embodiments, each cycle is about 21 days. In some embodiments, the pharmaceutical composition is administered at about 5 mg to about 25 mg (e.g., about 10 mg to about 25 mg, e.g., about 20 mg) of G-CSF dimer per administration. In some embodiments, the pharmaceutical composition is administered subcutaneously. In some embodiments, the pharmaceutical composition is packaged (e.g., prefilled) in a syringe (e.g., sterile and / or single-use). In some embodiments, the volume of the pharmaceutical composition in the syringe is about 1 mL.

[0210] In some embodiments, a method is provided for treating cancer (e.g., breast cancer) in an individual (e.g., a human), comprising administering to the individual an effective amount of a chemotherapeutic agent and further administering an effective amount of any of the pharmaceutical compositions described herein (e.g., a G-CSF-Fc dimer pharmaceutical composition, such as an efebmarenograstim alfa pharmaceutical composition) following (e.g., at least 24 hours after) administration of the chemotherapeutic agent to the individual, wherein administration of the pharmaceutical composition alleviates or prevents a condition characterized by impaired white blood cell production (e.g., chemotherapy-induced neutropenia or an infection). In some embodiments, the condition characterized by impaired white blood cell production is chemotherapy-induced neutropenia. In some embodiments, the chemotherapeutic agent is a myelosuppressive chemotherapeutic agent. In some embodiments, the myelosuppressive chemotherapeutic agent is selected from the group consisting of epirubicin, docetaxel, cyclophosphamide, doxorubicin, etoposide, cisplatin, paclitaxel, topotecan, vincristine, methylprednisolone, cytarabine, and combinations thereof. In some embodiments, the individual is administered two or more chemotherapeutic agents including: i) epirubicin and cyclophosphamide, ii) docetaxel and cyclophosphamide, iii) doxorubicin and cyclophosphamide, iv) docetaxel and doxorubicin, or v) docetaxel, doxorubicin, and cyclophosphamide. In some embodiments, the two or more chemotherapeutic agents are administered simultaneously (e.g., in the same formulation or in separate formulations). In some embodiments, the two or more chemotherapeutic agents are administered sequentially. In some embodiments, the cancer is selected from the group consisting of breast cancer, non-small cell lung cancer, small cell lung cancer, ovarian cancer, sarcoma, urothelial carcinoma, germ cell tumors, and non-Hodgkin's lymphoma. In some embodiments, administration of the G-CSF dimer pharmaceutical composition increases (e.g., by at least about 10%, 20%, 50%, 70%, 90%, 1-fold, 2-fold, 5-fold, 20-fold, or more) one or more of: i) ANC; ii) granulocyte count (e.g., in subjects eligible for bone marrow transplantation); iii) stem cell production; iv) hematopoiesis; and v) HPC count in an individual.In some embodiments, administration of the G-CSF dimer pharmaceutical composition reduces (e.g., by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more) the duration of chemotherapy-induced neutropenia (e.g., Grade 4) in an individual. In some embodiments, administration of the G-CSF dimer pharmaceutical composition reduces (e.g., by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more) or prevents the occurrence ...0.5 x 10) 9 In some embodiments, administration of the G-CSF dimer pharmaceutical composition reduces the duration of an ANC of less than about 0.5×10 / L to less than about 36 hours (e.g., less than about any of 34, 32, 30, 28, 26, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.5, 0.2, 0.1 hours or less). 9 / L (e.g., at least about 0.4 × 10 9 / L, 0.2 × 10 9 / L, 0.1 × 10 9 / L, 1.0 × 10 7 / L, or less, whichever is less. In some embodiments, administration of the G-CSF dimer pharmaceutical composition prevents the ANC from reaching about 0.5 x 10 9 / L or less within about 10 days (e.g., within about 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.2, 0.1 days, or less) of the first occurrence of 9 / L or more. In some embodiments, administration of the G-CSF dimer pharmaceutical composition reduces (e.g., reduces by at least about any of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more) or prevents the occurrence of FN in the individual. In some embodiments, administration of the G-CSF dimer pharmaceutical composition activates one or more of i) neutrophil progenitor cells, ii) bone marrow stem cells, and iii) mature neutrophils in the individual.In some embodiments, the method of treating cancer involves the production of the following biological activities: (1) killing of cancer cells (e.g., at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%); (2) inhibition of cancer cell proliferation (e.g., at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%); (3) reduction in tumor size (e.g., a decrease in tumor size); (4) a reduction (e.g., by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) of one or more symptoms in an individual with cancer; (5) a reduction (e.g., by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) of tumor metastasis (e.g., metastasis to lymph nodes); (6) a reduction (e.g., by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) in the development, occurrence, or burden of existing tumor metastasis (e.g., lymph node metastasis); (7) an increase in survival, e.g., at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, or 48 months of survival of an individual; or more, (8) extending the time to cancer progression, e.g., by at least about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 60 weeks or more, and (9) preventing, inhibiting, or reducing (e.g., by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) the likelihood of cancer recurrence.In some embodiments, the pharmaceutical composition comprises (or consists essentially of, or consists of) (a) about 20 mM G-CSF dimer (e.g., G-CSF-Fc dimer, e.g., efvemarenograstim alfa), (b) about 10 mM sodium acetate, (c) about 1 mM EDTA, (d) about 5% (w / v) sorbitol, and (e) about 0.01% (w / v) polysorbate 20, and the pH of the pharmaceutical composition is about 5.2. In some embodiments, the G-CSF dimers each comprise (or consist of) the amino acid sequence of any of SEQ ID NOS: 6-10 (e.g., SEQ ID NO: 6). In some embodiments, the G-CSF dimer comprises two monomeric subunits (consisting essentially of, or consisting of). In some embodiments, the G-CSF dimer is efvemarenograstim alfa. In some embodiments, the pharmaceutical composition is administered at about 5 mg to about 25 mg (e.g., about 10 mg to about 25 mg, e.g., about 20 mg) of G-CSF dimer per administration. In some embodiments, the pharmaceutical composition is administered subcutaneously. In some embodiments, the pharmaceutical composition is packaged (e.g., prefilled) in a syringe (e.g., sterile and / or single-use). In some embodiments, the volume of the pharmaceutical composition in the syringe is about 1 mL.

[0211] The G-CSF dimer (e.g., G-CSF-Fc dimer, e.g., efvemarenograstim alfa) pharmaceutical composition can be administered after administration of a chemotherapeutic agent to an individual, e.g., at least 24 hours after administration of the chemotherapeutic agent. In some embodiments, the pharmaceutical composition is administered 24, 28, 30, 32, 36, 40, 44, 48, 52, 60, 72, or more hours after administration of the chemotherapeutic agent, e.g., 24 or 48 hours after administration of the chemotherapeutic agent.

[0212] In some embodiments, when an individual receives two or more cycles (e.g., four cycles) of a chemotherapy agent, the administration of the G-CSF dimer pharmaceutical composition is after (e.g., at least 24 hours after) the administration of the last dose of the chemotherapy agent in the cycle. For example, when a chemotherapy agent is administered in one dose on day 1 of the cycle, the administration of the G-CSF dimer pharmaceutical composition is after (e.g., at least 24 hours after) the administration of the first dose of the chemotherapy agent in the cycle. When a chemotherapy agent is administered in a first dose on day 1 and a second dose on day 2 of the cycle (i.e., a cycle of at least two days), the administration of the G-CSF dimer pharmaceutical composition is after (e.g., at least 24 hours after) the administration of the second dose of the chemotherapy agent in the cycle. Similarly, when an individual receives two or more (e.g., four) doses of a chemotherapy agent in only one cycle, the administration of the G-CSF dimer pharmaceutical composition is after (e.g., at least 24 hours after) the administration of the last dose of the chemotherapy agent. In some embodiments, when an individual is administered two or more cycles (e.g., four cycles) of a chemotherapy agent, the administration of the G-CSF dimer pharmaceutical composition after (e.g., at least 24 hours after) the administration of the chemotherapy agent is simultaneous in each cycle (e.g., 24 hours or 48 hours after the administration of the chemotherapy agent in each cycle). In some embodiments, when an individual is administered two or more cycles (e.g., four cycles) of a chemotherapy agent, the administration of the G-CSF dimer pharmaceutical composition after (e.g., at least 24 hours after) the administration of the chemotherapy agent may be different in two or more cycles (e.g., 24 hours after the administration of the chemotherapy agent in one cycle and 24 hours after the administration of the chemotherapy agent in another cycle). In some embodiments, when an individual is administered two or more cycles (e.g., four cycles) of a chemotherapy agent, the administration of the G-CSF dimer pharmaceutical composition is after (e.g., at least 24 hours after) the administration of the chemotherapy agent in at least cycle 1 (e.g., cycle 1, cycles 1-4, or all cycles).

[0213] In some embodiments, the individual is administered two or more chemotherapeutic agents (e.g., docetaxel and cyclophosphamide), e.g., two or more chemotherapeutic agents per cycle. In some embodiments, the two or more chemotherapeutic agents are administered simultaneously (in the same formulation or in separate formulations). In some embodiments, the two or more chemotherapeutic agents are administered sequentially, e.g., a first chemotherapeutic agent is administered on day 1 and a second chemotherapeutic agent is administered on day 2. When an individual is administered two or more chemotherapeutic agents sequentially, in some embodiments, administration of the G-CSF dimer pharmaceutical composition is after (e.g., at least 24 hours after) administration of the last chemotherapeutic agent, e.g., the last chemotherapeutic agent of the cycle. In some embodiments, when two or more chemotherapeutic agents are administered at least two days apart, e.g., when a first chemotherapeutic agent is administered on day 1 and a second chemotherapeutic agent is administered on day 3 or later, depending on the ANC level and / or other condition (e.g., infection, fever, vital signs, clinical test values) of the individual being treated, the G-CSF dimer pharmaceutical composition can be administered after (e.g., at least 24 hours after) the administration of the first chemotherapeutic agent, or the G-CSF dimer pharmaceutical composition can be administered after (e.g., at least 24 hours after) the administration of each of the two or more chemotherapeutic agents.

[0214] In some embodiments, the individual is administered at least two cycles (e.g., two, three, or four cycles) of chemotherapy. In some embodiments, the G-CSF dimer pharmaceutical composition is administered after (e.g., at least 24 hours after) administration of the chemotherapy in cycle 1. In some embodiments, the individual is administered at least four cycles of chemotherapy, and the G-CSF dimer pharmaceutical composition is administered after (e.g., at least 24 hours after) administration of the chemotherapy in cycle 1. Thus, in some embodiments, methods are provided for treating or preventing a condition characterized by deficiency in white blood cell production (e.g., chemotherapy-induced neutropenia or infection) in an individual (e.g., a human, e.g., a human with cancer) in need thereof, wherein the individual has been administered at least two (e.g., 2, 3, or 4) cycles of a chemotherapy agent (e.g., the chemotherapy agent is administered on day 1 of each cycle), the method comprising administering to the individual an effective amount of any of the pharmaceutical compositions described herein (e.g., a G-CSF-Fc dimer pharmaceutical composition, e.g., an efvemarenograstim alfa pharmaceutical composition) after (e.g., at least 24 hours after) administration of cycle 1 of the chemotherapy agent. In some embodiments, provided are methods of treating or preventing a condition characterized by deficiency in white blood cell production (e.g., chemotherapy-induced neutropenia or infection) in an individual (e.g., a human, e.g., a human with cancer) in need thereof, where the individual has been administered at least four (e.g., 4) cycles of a chemotherapy agent (e.g., a chemotherapy agent is administered on day 1 of each cycle), comprising administering to the individual an effective amount of any of the pharmaceutical compositions described herein (e.g., a G-CSF-Fc dimer pharmaceutical composition, e.g., an efvemarenograstim alfa pharmaceutical composition) after (e.g., at least 24 hours after) administration of cycle 1 of the chemotherapy agent. In some embodiments, the individual is administered a chemotherapy agent for treating cancer (e.g., breast cancer).In some embodiments, provided are methods for treating cancer (e.g., breast cancer) in an individual (e.g., a human), comprising: i) administering to the individual an effective amount of a chemotherapeutic agent for at least two (e.g., 2, 3, or 4) cycles (e.g., the chemotherapeutic agent is administered on day 1 of each cycle); and ii) administering to the individual an effective amount of any of the pharmaceutical compositions described herein (e.g., a G-CSF-Fc dimer pharmaceutical composition, e.g., an efebmarenograstim alfa pharmaceutical composition) following administration of the chemotherapeutic agent to the individual in cycle 1 (e.g., at least 24 hours), wherein administration of the pharmaceutical composition alleviates or prevents a condition characterized by impaired white blood cell production (e.g., chemotherapy-induced neutropenia or infection) in at least one of the two or more cycles (e.g., cycle 1, or cycles 1-2). In some embodiments, a method is provided for treating cancer (e.g., breast cancer) in an individual (e.g., a human), comprising: i) administering to the individual an effective amount of a chemotherapeutic agent for at least four cycles (e.g., 4 cycles) (e.g., the chemotherapeutic agent is administered on day 1 of each cycle); and ii) administering an effective amount of any of the pharmaceutical compositions described herein (e.g., a G-CSF-Fc dimer pharmaceutical composition, e.g., an efebmarenograstim alfa pharmaceutical composition) after (e.g., at least 24 hours after) administration of the chemotherapeutic agent to the individual in cycle 1, wherein administration of the pharmaceutical composition alleviates or prevents a condition characterized by impaired white blood cell production (e.g., chemotherapy-induced neutropenia or infection) in at least one of the four or more cycles (e.g., cycle 1, or cycles 1-4). In some embodiments, the G-CSF dimer pharmaceutical composition is administered at least 24 hours (e.g., 24 hours, or 48 hours) after administration of the chemotherapeutic agent in cycle 1. In some embodiments, the method further comprises administering an effective amount of a G-CSF dimer pharmaceutical composition 24 hours (e.g., 24 hours, or any of 26, 28, 30, 32, 36, 40, 44, 48, 60, or more hours) after administration of the chemotherapeutic agent in each cycle, starting with cycle 2, e.g., in at least each of cycles 2 through 4.In some embodiments, the G-CSF dimer pharmaceutical composition is administered 48 hours (e.g., 48 hours or 48 hours) after administration of the chemotherapeutic agent in each cycle starting with cycle 2, e.g., in at least cycles 2-4. In some embodiments, the chemotherapeutic agent is administered on day 1 of each cycle (e.g., two or more days per cycle). In some embodiments, each cycle is about 21 days long. In some embodiments, the administration time of the G-CSF dimer pharmaceutical composition is the same in each cycle starting with cycle 2, e.g., in at least cycles 2-4. In some embodiments, the administration time of the G-CSF dimer pharmaceutical composition is different in two or more cycles starting with cycle 2, e.g., different in two or more cycles of at least cycles 2-4. In some embodiments, the condition characterized by impaired white blood cell production is chemotherapy-induced neutropenia. In some embodiments, the chemotherapeutic agent is a myelosuppressive chemotherapeutic agent. In some embodiments, two chemotherapeutic agents are administered to an individual in each cycle, e.g., simultaneously (e.g., in the same formulation or in separate formulations). In some embodiments, the pharmaceutical composition comprises (or consists essentially of, or consists of) (a) about 20 mM G-CSF dimer (e.g., G-CSF-Fc dimer, e.g., efvemarenograstim alfa), (b) about 10 mM sodium acetate, (c) about 1 mM EDTA, (d) about 5% (w / v) sorbitol, and (e) about 0.01% (w / v) polysorbate 20, and the pH of the pharmaceutical composition is about 5.2. In some embodiments, the G-CSF dimer is , each comprising two monomeric subunits comprising (or consisting essentially of, or consisting of) the amino acid sequence of any of SEQ ID NOS: 6-10 (e.g., SEQ ID NO: 6). In some embodiments, the G-CSF dimer is efvemarenograstim alfa. In some embodiments, the pharmaceutical composition is administered at about 5 mg to about 25 mg (e.g., about 10 mg to about 25 mg, e.g., about 20 mg) of G-CSF dimer per administration. In some embodiments, the pharmaceutical composition is administered subcutaneously.In some embodiments, the pharmaceutical composition is packaged (e.g., pre-filled) in a syringe (e.g., sterile and / or single-use). In some embodiments, the volume of the pharmaceutical composition in the syringe is about 1 mL. In some embodiments, administration of the G-CSF dimer pharmaceutical composition is effective in one or more cycles (e.g., cycle 1, cycles 1-2, cycles 2-4, or cycles 1-4) to achieve the following effects: i) a reduction in the duration of chemotherapy-induced neutropenia; ii) a reduction or prevention of the occurrence of chemotherapy-induced neutropenia; iii) a reduction or prevention of the occurrence of FN; iv) an increase in ANC; v) activation of neutrophil progenitor cells; vi) activation of bone marrow stem cells; vii) activation of mature neutrophils; viii) an increase in the number of neutrophils in an individual of about 0.5 x 10 9 / L to less than about 36 hours; ix) the individual's ANC is less than about 0.5 × 10 9 / L, x) ANC of approximately 0.5 × 10 9 / L within about 10 days of the first occurrence of 9 / L or greater, and xi) increasing the survival time of the individual. In some embodiments, the chemotherapy-induced neutropenia is Grade 4 neutropenia.

[0215] In some embodiments, the individual is administered at least two cycles (e.g., two, three, or four cycles) of a chemotherapy agent, and the G-CSF dimer pharmaceutical composition is administered after (e.g., at least 24 hours after) administration of the chemotherapy agent in each of the at least two cycles. In some embodiments, the individual is administered at least or four cycles of a chemotherapy agent, and the G-CSF dimer pharmaceutical composition is administered after (e.g., at least 24 hours after) administration of the chemotherapy agent in each of the at least four cycles. Thus, in some embodiments, methods are provided for treating or preventing a condition characterized by deficiency in white blood cell production (e.g., chemotherapy-induced neutropenia or infection) in an individual (e.g., a human, e.g., a human with cancer) in need thereof, wherein the individual has been administered at least two (e.g., 2, 3, or 4) cycles of a chemotherapy agent (e.g., the chemotherapy agent is administered on day 1 of each cycle), the method comprising administering to the individual an effective amount of any of the pharmaceutical compositions described herein (e.g., a G-CSF-Fc dimer pharmaceutical composition, e.g., an efvemarenograstim alfa pharmaceutical composition) after (e.g., at least 24 hours after) administration of the chemotherapy agent in each of the at least two cycles. Thus, in some embodiments, provided are methods of treating or preventing a condition characterized by deficiency in white blood cell production (e.g., chemotherapy-induced neutropenia or infection) in an individual (e.g., a human, e.g., a human with cancer) in need thereof, where the individual has been administered at least four cycles (e.g., four cycles) of a chemotherapy agent (e.g., the chemotherapy agent is administered on day 1 of each cycle), the method comprising administering to the individual an effective amount of any of the pharmaceutical compositions described herein (e.g., a G-CSF-Fc dimer pharmaceutical composition, e.g., an efvemarenograstim alfa pharmaceutical composition) after (e.g., at least 24 hours after) administration of the chemotherapy agent for each of the at least four cycles. In some embodiments, the individual is administered a chemotherapy agent for treating cancer (e.g., breast cancer).In some embodiments, provided are methods for treating cancer (e.g., breast cancer) in an individual (e.g., a human, e.g., an individual having cancer), comprising: i) administering to the individual an effective amount of a chemotherapeutic agent for at least two (e.g., 2, 3, or 4) cycles (e.g., the chemotherapeutic agent is administered on day 1 of each cycle); and ii) administering an effective amount of any of the pharmaceutical compositions described herein (e.g., a G-CSF-Fc dimer pharmaceutical composition, e.g., an efebmarenograstim alfa pharmaceutical composition) following administration of the chemotherapeutic agent to the individual in each of the at least two cycles (e.g., at least 24 hours), wherein administration of the pharmaceutical composition alleviates or prevents a condition characterized by impaired white blood cell production (e.g., chemotherapy-induced neutropenia or infection) in at least one of the two or more cycles (e.g., cycle 1, or cycles 1-2). In some embodiments, a method is provided for treating cancer (e.g., breast cancer) in an individual (e.g., a human, e.g., an individual with cancer), comprising: i) administering to the individual an effective amount of a chemotherapeutic agent for at least four cycles (e.g., 4 cycles) (e.g., the chemotherapeutic agent is administered on day 1 of each cycle); and ii) administering an effective amount of any of the pharmaceutical compositions described herein (e.g., a G-CSF-Fc dimer pharmaceutical composition, e.g., an efebmarenograstim alfa pharmaceutical composition) after (e.g., at least 24 hours after) administration of the chemotherapeutic agent to the individual in each of the at least four cycles, wherein administration of the pharmaceutical composition alleviates or prevents a condition characterized by impaired white blood cell production (e.g., chemotherapy-induced neutropenia or infection) in at least one of the four or more cycles (e.g., cycle 1, or cycles 1-4). In some embodiments, the chemotherapeutic agent is administered on day 1 of each cycle (e.g., two or more days per cycle). In some embodiments, each cycle is about 21 days long. In some embodiments, the administration time of the G-CSF dimer pharmaceutical composition is the same for each of the at least two cycles. In some embodiments, the administration time of the G-CSF dimer pharmaceutical composition is different for each of the at least two cycles.In some embodiments, the administration time of the G-CSF dimer pharmaceutical composition is the same in each of the at least four cycles. In some embodiments, the administration time of the G-CSF dimer pharmaceutical composition is different in two or more (e.g., four) of the at least four cycles. In some embodiments, the G-CSF dimer pharmaceutical composition is administered at least 24 hours (e.g., 24 hours or 48 hours) after administration of the chemotherapeutic agent in each of at least two cycles, e.g., in each of at least four cycles. In some embodiments, the condition characterized by impaired white blood cell production is chemotherapy-induced neutropenia. In some embodiments, the chemotherapeutic agent is a myelosuppressive chemotherapeutic agent. In some embodiments, two chemotherapeutic agents are administered to an individual in each cycle, for example, simultaneously (e.g., in the same formulation or in separate formulations). In some embodiments, the pharmaceutical composition comprises (or consists essentially of, or consists of) (a) about 20 mM G-CSF dimer (e.g., G-CSF-Fc dimer, e.g., efvemarenograstim alfa), (b) about 10 mM sodium acetate, (c) about 1 mM EDTA, (d) about 5% (w / v) sorbitol, and (e) about 0.01% (w / v) polysorbate 20, and the pH of the pharmaceutical composition is about 5.2. In some embodiments, the G-CSF dimers each comprise (or consist of) the amino acid sequence of any of SEQ ID NOS: 6-10 (e.g., SEQ ID NO: 6). In some embodiments, the G-CSF dimer comprises two monomeric subunits (consisting essentially of, or consisting of). In some embodiments, the G-CSF dimer is efvemarenograstim alfa. In some embodiments, the pharmaceutical composition is administered at about 5 mg to about 25 mg (e.g., about 10 mg to about 25 mg, e.g., about 20 mg) of G-CSF dimer per administration. In some embodiments, the pharmaceutical composition is administered subcutaneously. In some embodiments, the pharmaceutical composition is packaged (e.g., prefilled) in a syringe (e.g., sterile and / or single-use). In some embodiments, the volume of the pharmaceutical composition in the syringe is about 1 mL.In some embodiments, administration of the G-CSF dimer pharmaceutical composition, in one or more cycles (e.g., Cycle 1, Cycles 1-2, Cycles 2-4, or Cycles 1-4), achieves the following effects: i) a reduction in the duration of chemotherapy-induced neutropenia, ii) a reduction or prevention of the occurrence of chemotherapy-induced neutropenia, iii) a reduction or prevention of the occurrence of FN, iv) an increase in ANC, v) activation of neutrophil progenitor cells, vi) activation of bone marrow stem cells, vii) activation of mature neutrophils, viii) an increase in the number of neutrophils in about 0.5 x 10 individuals. 9 / L to less than about 36 hours; ix) the individual's ANC is less than about 0.5 × 10 9 / L, x) ANC of approximately 0.5 × 10 9 / L within about 10 days of the first occurrence of 9 / L or greater, and xi) increasing the survival time of the individual. In some embodiments, the chemotherapy-induced neutropenia is Grade 4 neutropenia.

[0216] In some embodiments, a method for treating or preventing a condition characterized by impaired white blood cell production (e.g., chemotherapy-induced neutropenia or an infection) in an individual (e.g., a human) in need thereof, comprising: administering an effective amount of any of the pharmaceutical compositions described herein (e.g., a G-CSF-Fc dimer pharmaceutical composition, e.g., an efvemarenograstim alfa pharmaceutical composition) after administration of one or more chemotherapy agents in each cycle of at least four cycles, wherein the individual has breast cancer (e.g., metastatic or non-metastatic), and (i) the individual has been administered at least four cycles of docetaxel and cyclophosphamide, and 2 and cyclophosphamide 600 mg / m 2 is administered on day 1 of each cycle and the pharmaceutical composition is administered on day 2 of each cycle at least 24 hours after the administration of docetaxel and cyclophosphamide, or (ii) the individual is administered at least four cycles of epirubicin and cyclophosphamide and epirubicin 100 mg / m 2 and cyclophosphamide 600 mg / m2 is administered on day 1 of each cycle, and the pharmaceutical composition is administered on day 3 of each cycle, at least 48 hours after the administration of epirubicin and cyclophosphamide; or (iii) the individual is administered at least four cycles of docetaxel and doxorubicin, and docetaxel 75 mg / m 2 and doxorubicin 60 mg / m 2 is administered on day 1 of each cycle, and the pharmaceutical composition is administered on day 2 of each cycle, at least 24 hours after the administration of docetaxel and doxorubicin. In some embodiments, a method for treating breast cancer (e.g., metastatic or non-metastatic) in an individual (e.g., a human) in need thereof, comprising administering one or more chemotherapeutic agents in each cycle of at least four cycles, and further administering an effective amount of any of the pharmaceutical compositions described herein (e.g., a G-CSF-Fc dimer pharmaceutical composition, e.g., an efvemarenograstim alfa pharmaceutical composition) after the administration of the one or more chemotherapeutic agents in each cycle of the at least four cycles, wherein administration of the pharmaceutical composition alleviates or prevents a condition characterized by impaired white blood cell production (e.g., chemotherapy-induced neutropenia or infection), wherein (i) the individual is administered at least four cycles of docetaxel and cyclophosphamide, and 2 and cyclophosphamide 600 mg / m 2 is administered on day 1 of each cycle and the pharmaceutical composition is administered on day 2 of each cycle at least 24 hours after the administration of docetaxel and cyclophosphamide, or (ii) the individual is administered at least four cycles of epirubicin and cyclophosphamide and epirubicin 100 mg / m 2 and cyclophosphamide 600 mg / m 2 is administered on day 1 of each cycle, and the pharmaceutical composition is administered on day 3 of each cycle, at least 48 hours after the administration of epirubicin and cyclophosphamide; or (iii) the individual is administered at least four cycles of docetaxel and doxorubicin, and docetaxel 75 mg / m 2 and doxorubicin 60 mg / m 2is administered on day 1 of each cycle, and the pharmaceutical composition is administered on day 2 of each cycle at least 24 hours after the administration of docetaxel and doxorubicin. In some embodiments, each cycle is about 21 days. In some embodiments, the pharmaceutical composition comprises (or consists essentially of, or consists of) (a) about 20 mM G-CSF dimer (e.g., a G-CSF-Fc dimer, e.g., efvemarenograstim alfa), (b) about 10 mM sodium acetate, (c) about 1 mM EDTA, (d) about 5% (w / v) sorbitol, and (e) about 0.01% (w / v) polysorbate 20, and the pH of the pharmaceutical composition is about 5.2. In some embodiments, the G-CSF dimers each comprise (or consist of) the amino acid sequence of any of SEQ ID NOS: 6-10 (e.g., SEQ ID NO: 6). In some embodiments, the G-CSF dimer comprises two monomeric subunits (consisting essentially of, or consisting of). In some embodiments, the G-CSF dimer is efvemarenograstim alfa. In some embodiments, the pharmaceutical composition is administered at about 5 mg to about 25 mg (e.g., about 10 mg to about 25 mg, e.g., about 20 mg) of G-CSF dimer per administration. In some embodiments, the pharmaceutical composition is administered subcutaneously. In some embodiments, the pharmaceutical composition is packaged (e.g., prefilled) in a syringe (e.g., sterile and / or single-use). In some embodiments, the volume of the pharmaceutical composition in the syringe is about 1 mL.

[0217] In some embodiments, the methods described herein, or administration of a G-CSF dimer pharmaceutical composition (e.g., a G-CSF-Fc dimer pharmaceutical composition, e.g., an efvemarenograstim alfa pharmaceutical composition) after (e.g., at least 24 hours after) administration of a chemotherapeutic agent to an individual, induces an increase in white blood cells (WBCs) (e.g., an increase of at least about any of a 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 1-fold, 2-fold, 5-fold, 10-fold, 50-fold, or more) or a decrease in loss of WBCs (e.g., a decrease of at least about any of a 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more) in the individual. For example, in some embodiments, neutrophil count (e.g., ANC) is increased in an individual (e.g., by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 1-fold, 2-fold, 5-fold, 10-fold, 20-fold, or more). In some embodiments, a decrease in neutrophil count is inhibited in an individual (e.g., by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 1-fold, 2-fold, 5-fold, 10-fold, or more). In some embodiments, nadir neutrophil count is increased in an individual (e.g., by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 1-fold, 2-fold, 5-fold, 10-fold, or more). In some embodiments, the recovery neutrophil count is increased in the individual (e.g., by at least about any of a 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 1-fold, 2-fold, 5-fold, 10-fold, or more increase). In some embodiments, the time to ANC recovery is decreased in the individual (e.g., by at least about any of a 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more decrease). In some embodiments, one or more of the effects described herein are achieved in the individual's first cycle of chemotherapy.In some embodiments, one or more of the benefits described herein are achieved in one or more cycles of chemotherapy in an individual, e.g., cycles 1-2 of two or more cycles, or cycles 1-3 of four or more cycles. In some embodiments, one or more of the benefits described herein are achieved in each cycle (e.g., all two cycles, or all four cycles) of chemotherapy in an individual.

[0218] In some embodiments, the methods described herein, or administration of a G-CSF dimer pharmaceutical composition (e.g., a G-CSF-Fc dimer pharmaceutical composition, e.g., an efvemarenograstim alfa pharmaceutical composition) after (e.g., at least 24 hours after) administration of a chemotherapeutic agent to an individual, reduces (e.g., by at least about any of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more) the duration of chemotherapy-induced neutropenia (e.g., grade 4) in the individual. In some embodiments, the duration of chemotherapy-induced neutropenia is reduced by one or more grades of neutropenia, e.g., grade I, grade II, grade III, or grade IV. In some embodiments, the methods described herein reduce the duration of grade 4 (severe) neutropenia, i.e., by about 0.5×10 9 / L to less than about 36 hours (e.g., less than about any of 34, 32, 30, 28, 26, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.1 hours or less). In some embodiments, the methods described herein reduce the duration of Grade 4 (severe) neutropenia, or to less than about 0.5 x 10 9 / L to less than about 36 hours. In some embodiments, grade 4 (severe) neutropenia (ANC < 0.5 x 10 9 / L) is the duration of time an individual has an ANC <0.5 × 10 in cycle 1 of the individual's chemotherapy. 9 / L. In some embodiments, the methods described herein reduce the duration of Grade 4 neutropenia or reduce the number of days an individual has Grade 4 neutropenia by about 0.5 x 10 / L in cycle 1 of chemotherapy. 9 In some embodiments, the methods described herein reduce the duration of Grade 4 neutropenia or reduce the duration of ANC below about 0.5×10 / L in one or more cycles of chemotherapy in an individual, e.g., cycles 1-2 of two or more cycles, or cycles 1-3 of four or more cycles. 9 In some embodiments, the methods described herein reduce the duration of Grade 4 neutropenia or reduce the duration of ANC below about 0.5×10 / L in each cycle (e.g., all 2 cycles, or all 4 cycles) of chemotherapy in an individual. 9 / L. In some embodiments, the methods described herein reduce the duration of Grade 3 neutropenia, i.e., about 1.0 x 10 9 / L to less than about 36 hours (e.g., less than about 34, 32, 30, 28, 26, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.1 hours, or less). In some embodiments, grade 3 neutropenia (ANC<1.0×10) is reduced to less than about 36 hours (e.g., less than about 34, 32, 30, 28, 26, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.1 hours, or less). 9 / L) is the duration of time an individual has an ANC < 1.0 × 10 in cycle 1 of the individual's chemotherapy. 9 / L. In some embodiments, the methods described herein reduce the duration of Grade 3 neutropenia or reduce the number of days an individual has had Grade 3 neutropenia by about 1.0 x 10 / L in cycle 1 of chemotherapy. 9 In some embodiments, the methods described herein reduce the duration of Grade 3 neutropenia or reduce the duration of ANC below about 1.0 x 10 / L in one or more cycles of chemotherapy in an individual, e.g., cycles 1-2 for two or more cycles, or cycles 1-3 for four or more cycles. 9In some embodiments, the methods described herein reduce the duration of Grade 3 neutropenia or reduce the duration of ANC below about 1.0 x 10 / L in each cycle (e.g., all 2 cycles, or all 4 cycles) of chemotherapy in an individual. 9 Decrease the duration of ANC below / L.

[0219] In some embodiments, the methods described herein, or administration of a G-CSF dimer pharmaceutical composition (e.g., a G-CSF-Fc dimer pharmaceutical composition, e.g., an efvemarenograstim alfa pharmaceutical composition) after (e.g., at least 24 hours after) administration of a chemotherapeutic agent to an individual, reduces (e.g., by at least about any of a 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more reduction) or prevents the occurrence of chemotherapy-induced neutropenia (e.g., grade 4) in the individual. In some embodiments, the occurrence of chemotherapy-induced neutropenia is reduced by one or more grades of neutropenia, e.g., grade I, grade II, grade III, or grade IV. In some embodiments, the occurrence of chemotherapy-induced neutropenia is reduced or prevented for any grade of neutropenia. In some embodiments, the methods described herein reduce or prevent the occurrence of grade 4 (severe) neutropenia. In some embodiments, the methods described herein provide for an ANC in an individual of about 0.5×10 9 / L. In some embodiments, the methods described herein reduce the incidence of Grade 4 neutropenia or reduce the incidence of Grade 5 neutropenia to about 0.5 x 10 9 / L by at least one, e.g., at least two, three, four, or more times. In some embodiments, the methods described herein reduce or prevent the occurrence of Grade 4 neutropenia or a reduction in the incidence of Grade 5 neutropenia of about 0.5 x 10 / L in an individual during Cycle 1 of chemotherapy. 9In some embodiments, the methods described herein reduce or prevent the occurrence of Grade 4 neutropenia or an ANC of less than about 0.5 x 10 / L in one or more cycles of chemotherapy in an individual, e.g., cycles 1-2 of two or more cycles, or cycles 1-3 of four or more cycles. 9 In some embodiments, the methods described herein reduce or prevent the occurrence of Grade 4 neutropenia or an ANC of less than about 0.5 x 10 / L in each cycle (e.g., all 2 cycles, or all 4 cycles) of chemotherapy in an individual. 9 In some embodiments, the methods described herein reduce or prevent the occurrence of Grade 3 neutropenia. In some embodiments, the methods described herein reduce or prevent the occurrence of Grade 3 neutropenia. In some embodiments, the methods described herein reduce or prevent the occurrence of Grade 3 neutropenia in an individual when the ANC reaches about 1.0 x 10 9 / L. In some embodiments, the methods described herein reduce the incidence of Grade 3 neutropenia or reduce the incidence of Grade 4 neutropenia to about 1.0 x 10 9 / L by at least one, e.g., at least two, three, four, or more times. In some embodiments, the methods described herein reduce or prevent the occurrence of Grade 3 neutropenia or a reduction in ANC of about 1.0 x 10 / L in an individual during Cycle 1 of chemotherapy. 9 In some embodiments, the methods described herein reduce or prevent the occurrence of Grade 3 neutropenia or an ANC of less than about 1.0 x 10 / L in one or more cycles of chemotherapy in an individual, e.g., cycles 1-2 of two or more cycles, or cycles 1-3 of four or more cycles. 9 In some embodiments, the methods described herein reduce or prevent the occurrence of Grade 3 neutropenia or an ANC of less than about 1.0 x 10 / L in each cycle (e.g., all 2 cycles, or all 4 cycles) of chemotherapy in an individual. 9 Reduce or prevent the occurrence of ANC reaching ANC less than / L.

[0220] In some embodiments, the methods described herein or administration of a G-CSF dimer pharmaceutical composition (e.g., a G-CSF-Fc dimer pharmaceutical composition, e.g., an efvemarenograstim alfa pharmaceutical composition) after (e.g., at least 24 hours after) administration of a chemotherapeutic agent to an individual reduces (e.g., by at least about any of a 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more reduction) or prevents the occurrence of febrile neutropenia (FN) in the individual. FN is defined as a single oral temperature of 38.3°C (101°F) or greater or a temperature greater than 38.0°C (100.4°F) sustained for at least one hour and an ANC of 0.5 x 10 or greater on the same day. 9 / L. Thus, in some embodiments, the methods described herein are directed to detecting a single oral temperature of 38.3°C (101°F) or greater that lasts for more than one hour or a temperature of greater than 38.0°C (100.4°F) in an individual and a blood glucose level of 0.5 x 10 / L on the same day (e.g., within about any of 24, 20, 18, 16, 14, 12, 10, 8, 6, 4, 2, 1, 0.5 hours, or less). 9 In some embodiments, the methods described herein reduce or prevent the occurrence of ANC less than 1 / L. In some embodiments, the methods described herein reduce the occurrence of FN at least once, e.g., at least 2, 3, 4, or more times. In some embodiments, the methods described herein reduce or prevent the occurrence of FN in cycle 1 of an individual's chemotherapy. In some embodiments, the methods described herein reduce or prevent the occurrence of FN in one or more cycles of an individual's chemotherapy, e.g., cycles 1-2 of two or more cycles, or cycles 1-3 of four or more cycles. In some embodiments, the methods described herein reduce or prevent the occurrence of FN in each cycle (e.g., all two cycles, or all four cycles) of an individual's chemotherapy.

[0221] In some embodiments, the methods described herein, or administration of a G-CSF dimer pharmaceutical composition (e.g., a G-CSF-Fc dimer pharmaceutical composition, e.g., an efvemarenograstim alfa pharmaceutical composition) after (e.g., at least 24 hours after) administration of a chemotherapeutic agent to an individual, reduces (e.g., by about any of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more) the duration and / or occurrence of, or prevents the occurrence of, an infection (e.g., an infection manifested by neutropenia, e.g., FN) in the individual. In some embodiments, the methods described herein reduce (e.g., by at least about any of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more) or prevents the duration and / or occurrence of intravenous antibiotic use. In some embodiments, the methods described herein reduce the occurrence of infection at least once, e.g., at least two, three, four, or more times. In some embodiments, the methods described herein reduce the duration and / or occurrence of, or prevent the occurrence of, an infection during cycle 1 of an individual's chemotherapy. In some embodiments, the methods described herein reduce the duration and / or occurrence of, or prevent the occurrence of, an infection during one or more cycles of an individual's chemotherapy, e.g., cycles 1-2 of two or more cycles, or cycles 1-3 of four or more cycles. In some embodiments, the methods described herein reduce the duration and / or occurrence of, or prevent the occurrence of, an infection during each cycle (e.g., all two cycles, or all four cycles) of an individual's chemotherapy.

[0222] In some embodiments, the methods described herein, or administration of a G-CSF dimer pharmaceutical composition (e.g., a G-CSF-Fc dimer pharmaceutical composition, e.g., an efvemarenograstim alfa pharmaceutical composition) after (e.g., at least 24 hours after) administration of a chemotherapeutic agent to an individual, reduces (e.g., by about any of a 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more) the duration and / or occurrence of febrile neutropenia or hospitalization for infection in the individual, or prevents its occurrence. In some embodiments, the methods described herein reduce (e.g., by at least about any of a 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more) or prevents the duration and / or occurrence of intravenous antibiotic use. In some embodiments, the methods described herein reduce the incidence of hospitalization for FN or infection by at least one time, e.g., at least two, three, four, or more times. In some embodiments, the methods described herein reduce the duration and / or incidence of, or prevent, hospitalization for FN or infection during cycle 1 of an individual's chemotherapy. In some embodiments, the methods described herein reduce the duration and / or incidence of, or prevent, hospitalization for FN or infection during one or more cycles of chemotherapy, e.g., cycles 1-2 of two or more cycles, or cycles 1-3 of four or more cycles, of an individual's chemotherapy. In some embodiments, the methods described herein reduce the duration and / or incidence of, or prevent hospitalization for FN or infection during each cycle (e.g., all two cycles, or all four cycles) of chemotherapy for an individual.

[0223] In some embodiments, the methods described herein, or administration of a G-CSF dimer pharmaceutical composition (e.g., a G-CSF-Fc dimer pharmaceutical composition, e.g., an efvemarenograstim alfa pharmaceutical composition) after (e.g., at least 24 hours after) administration of a chemotherapy agent to an individual, reduces the individual's ANC nadir depth (e.g., by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more) or increases the nadir value (e.g., by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 1-fold, 2-fold, 5-fold, 10-fold, or more). In some embodiments, the methods described herein reduce the individual's ANC nadir depth or increase the ANC nadir value in cycle 1 of chemotherapy. In some embodiments, the methods described herein reduce ANC nadir depth or increase ANC nadir value in one or more cycles of chemotherapy in an individual, e.g., cycles 1-2 of two or more cycles, or cycles 1-3 of four or more cycles. In some embodiments, the methods described herein reduce ANC nadir depth or increase ANC nadir value in each cycle (e.g., all two cycles, or all four cycles) of chemotherapy in an individual.

[0224] In some embodiments, the methods described herein, or administration of a G-CSF dimer pharmaceutical composition (e.g., a G-CSF-Fc dimer pharmaceutical composition, e.g., an efvemarenograstim alfa pharmaceutical composition) after (e.g., at least 24 hours after) administration of a chemotherapeutic agent to an individual, reduces the individual's time to ANC recovery (e.g., by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more). In some embodiments, the individual's time to ANC recovery is reduced by at least about 5 minutes, 30 minutes, 1 hour, 2 hours, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days, or more. In some embodiments, ANC recovery refers to an ANC recovery of 2.0×10 after the ANC nadir (e.g., the expected ANC nadir). 9 / L or greater. Thus, in some embodiments, the methods described herein can be used to measure the ANC nadir, e.g., about 0.5×10 9 After the first occurrence of ANC less than / L, an ANC of 2.0 × 10 9 In some embodiments, the methods described herein, or administration of a G-CSF dimer pharmaceutical composition (e.g., a G-CSF-Fc dimer pharmaceutical composition, e.g., an efvemarenograstim alfa pharmaceutical composition) after (e.g., at least 24 hours after) administration of a chemotherapeutic agent to an individual, reduces the ANC by about 0.5×10 9 / L, or less, within about 10 days, e.g., within about 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.2, 0.1 days, or less. 9 / L or greater. In some embodiments, the methods described herein increase the ANC to about 0.5 x 10 9 / L or less within about 10 days, e.g., about 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.2, 0.1 days, or less, of the first occurrence of 9 / L or more. In some embodiments, the methods described herein decrease the time to ANC recovery in cycle 1 of chemotherapy in an individual. In some embodiments, the methods described herein decrease the time to ANC recovery in one or more cycles of chemotherapy in an individual, e.g., cycles 1-2 of two or more cycles, or cycles 1-3 of four or more cycles. In some embodiments, the methods described herein decrease the time to ANC recovery in each cycle (e.g., all two cycles, or all four cycles) of chemotherapy in an individual.

[0225] In some embodiments, one or more effects (e.g., therapeutic effects) achieved by a method described herein or by administration of a G-CSF dimer pharmaceutical composition (e.g., a G-CSF-Fc dimer pharmaceutical composition, e.g., an efvemarenograstim alfa pharmaceutical composition) after (e.g., at least 24 hours after) administration of a chemotherapeutic agent to an individual are compared to an individual who is not administered a G-CSF dimer pharmaceutical composition (e.g., such as any of the G-CSF dimer pharmaceutical compositions described herein). In some embodiments, one or more effects achieved by the methods described herein are compared to an individual receiving pegfilgrastim (e.g., Neulasta®) or biosimilar, efrapegrastim (e.g., Rolontis®, HM10460A, or ROLVEDON™) or biosimilar, or filgrastim (GRAN®) or biosimilar on the same treatment schedule as the G-CSF dimer pharmaceutical composition, i.e., after administration of the same chemotherapeutic agent (e.g., 24 hours or 48 hours later). In some embodiments, one or more effects achieved by the methods described herein are compared to an individual administered pegfilgrastim (e.g., Neulasta®) or biosimilar, efrapegrastim (e.g., Rolontis®, HM10460A, or ROLVEDON™) or biosimilar, or filgrastim (GRAN®) or biosimilar, at a G-CSF dose equivalent to the G-CSF dimer pharmaceutical composition (e.g., the administration schedule may be different).In some embodiments, the methods of using the G-CSF dimer pharmaceutical compositions described herein i) achieve a similar (e.g., within about a 5% difference) or better therapeutic effect and / or ii) have similar (e.g., within about a 5% difference) or fewer adverse events compared to an individual administered pegfilgrastim (e.g., Neulasta®) or biosimilar, efrapegrastim (e.g., Rolontis®, HM10460A, or ROLVEDON™) or biosimilar, or filgrastim (GRAN®) or biosimilar, at the same treatment schedule and / or equivalent G-CSF dose as the G-CSF dimer pharmaceutical composition.

[0226] In some embodiments, the individual is a human. In some embodiments, the G-CSF dimer pharmaceutical composition (e.g., a G-CSF-Fc dimer pharmaceutical composition, e.g., an efvemarenograstim alfa pharmaceutical composition) is administered at about 5 mg to about 25 mg per administration, e.g., about 10 mg to about 25 mg, about 5 mg to about 15 mg, about 5 mg to about 20 mg, about 10 mg to about 15 mg, about 10 mg to about 20 mg, about 15 mg to about 25 mg, about 15 mg to about 20 mg, or about 18 mg to about 22 mg per administration. In some embodiments, the G-CSF dimer pharmaceutical composition is administered at about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 mg per administration, or any value therebetween. In some embodiments, the G-CSF dimer pharmaceutical composition is administered at about 20 mg per administration. In some embodiments, the individual is a non-human animal. In some embodiments, the G-CSF dimer pharmaceutical composition is administered to the non-human animal at a dose equivalent to a human dose. In some embodiments, when the individual is administered two or more cycles of chemotherapy, the G-CSF dimer pharmaceutical composition is administered in the same amount (e.g., 20 mg) in each cycle. In some embodiments, when the individual is administered two or more cycles of chemotherapy, the G-CSF dimer pharmaceutical composition is administered in different amounts in at least two of the cycles.

[0227] In some embodiments, the individual to be treated or administered the G-CSF dimer pharmaceutical composition (e.g., a G-CSF-Fc dimer pharmaceutical composition, e.g., an efvemarenograstim alfa pharmaceutical composition) meets one or more of the following criteria: i) for humans, at least 18 years of age; ii) for individuals with breast cancer (e.g., stage I-III breast cancer), female; iii) scheduled to receive two or more cycles (e.g., 2, 3, 4, or more) of chemotherapy (e.g., neoadjuvant or adjuvant); iv) Eastern Cooperative Oncology Group (ECOG) outcome status of 2 or less; v) ANC ≥ 2.0 x 10 before administration of chemotherapy. 9 / L, hemoglobin ≥ 11.0 g / dL, and platelet count ≥ 100 × 10 9 / L, vi) have adequate renal, hepatic, and cardiac function (e.g., alanine aminotransferase (ALT), aspartate aminotransferase (AST), and / or alkaline phosphatase less than 2.5 times the upper limit of normal (ULN); total bilirubin and / or serum creatinine less than 1.5 x ULN); vii) use of contraception for at least one month prior to and during administration of the chemotherapeutic agent. In some embodiments, the individual does not have a latex allergy. In some embodiments, the individual does not have a severe allergic reaction to efvemarenograstim alfa. In some embodiments, the methods described herein further comprise selecting an individual who meets one or more of the above criteria.

[0228] In some embodiments, the pharmaceutical composition is administered at a dose of at least about 0.01 mg / kg, e.g., at least about 0.02 mg / kg, 0.05 mg / kg, 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg, or 1 mg / kg. In some embodiments, the pharmaceutical composition is administered at a dose of about 0.01 mg / kg to about 1 mg / kg, e.g., about 0.02 mg / kg to about 0.9 mg / kg, about 0.05 mg / kg to about 0.8 mg / kg, or about 0.1 mg / kg to about 0.7 mg / kg. In some embodiments, the pharmaceutical composition is administered at a dose of about 0.01 mg / kg, about 0.02 mg / kg, about 0.05 mg / kg, about 0.1 mg / kg, about 0.2 mg / kg, about 0.3 mg / kg, about 0.4 mg / kg, about 0.5 mg / kg, about 0.6 mg / kg, about 0.7 mg / kg, about 0.8 mg / kg, about 0.9 mg / kg, or about 1 mg / kg.

[0229] The pharmaceutical composition can be administered at any desired regimen and frequency. In some embodiments of any of the methods described herein, the pharmaceutical composition is administered once a day, once every two days, once every three days, once every four days, once every five days, once every six days, once a week, once every two weeks, once every three weeks, once every four weeks, once every six weeks, or once every eight weeks. In some embodiments, the pharmaceutical composition is administered once every four weeks. In some embodiments, the pharmaceutical composition is administered at about 20 mg per administration, for example, about 20 mg per chemotherapy cycle. In some embodiments, the method comprises administering the pharmaceutical composition as a single dose.

[0230] In some embodiments, the pharmaceutical composition is administered using a syringe (e.g., a pre-filled syringe), for example, any of the syringes described herein. In some embodiments, the pharmaceutical composition is administered subcutaneously. In some embodiments, the pharmaceutical composition is contained in a syringe (e.g., pre-filled). In some embodiments, the volume of the pharmaceutical composition in the syringe is about 1 mL. In some embodiments, the syringe contains about 20 mg of G-CSF dimer, for example, any of the G-CSF dimers described herein (e.g., G-CSF-Fc dimer, e.g., efvemarenograstim alfa). In some embodiments, the syringe is sterile. In some embodiments, the syringe is for single use.

[0231] Pharmaceutical compositions can be administered to an individual (e.g., a human, e.g., an individual with cancer) via a variety of routes, including, for example, intravenous, intraarterial, intraperitoneal, intravascular, intramuscular, subcutaneous, transmucosal, and transdermal. In some embodiments, sustained-release formulations of the pharmaceutical composition can be used. In some embodiments, implantation of a sustained-release device, e.g., a mini-osmotic pump, can be used. Other delivery modes include, but are not limited to, the use of liposomal formulations or intravenous infusion. In some embodiments, the pharmaceutical composition is administered intravenously. In some embodiments, the pharmaceutical composition is administered intraperitoneally. In some embodiments, the pharmaceutical composition is administered intramuscularly. In some embodiments, the pharmaceutical composition is administered subcutaneously. When administered subcutaneously, such pharmaceutical compositions are typically administered in a volume of less than about 2.0 mL per injection site, e.g., about 1.5 mL, about 1 mL, or about 0.5 mL. In some embodiments, the injection volume is 1 mL.

[0232] Neutropenia Neutropenia is defined as a peripheral blood neutrophil count of 1.8 x 10 in adults. 9 / L, and 1.5 × 10 9 Neutropenia is often a precursor to infection, and the lower the neutrophil count, the higher the risk of infection.

[0233] Guidelines used to classify neutropenia are shown in Table A. The frequency and severity of infections caused by neutropenia are also influenced by other factors, such as the integrity of mucous membranes and skin, and the function and levels of immunoglobulins, lymphocytes, monocytes, and the complement system. [Table 2]

[0234] According to the cause of neutropenia, common clinical neutropenia can be classified into the following categories: impaired hematopoietic production caused by secondary factors such as drugs, radiation, chemical reagents, and infections; altered in vivo distribution and circulation; and increased utilization and turnover. The severity of chemotherapy-induced neutropenia in tumor patients generally depends on the chemotherapy dose, and repeated chemotherapy can have a cumulative effect on neutropenia. The main clinical consequence of neutropenia is infectious complications. Many of the infections in these patients are primarily caused by aerobic bacteria, including gram-negative bacteria (Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa), gram-positive bacteria (Staphylococcus aureus, alpha-hemolytic streptococci, and Staphylococcus aureus), and fungi.

[0235] Cytotoxic chemotherapy remains one of the main treatments for cancer. The greatest drawback of chemotherapy is that it kills rapidly proliferating and differentiating healthy cells indiscriminately from tumor cells. The toxicity caused by chemotherapy is primarily reflected in the hematopoietic system and is clinically known as chemotherapy-induced neutropenia.

[0236] Neutropenia may delay the next treatment cycle and directly affect the therapeutic efficacy of chemotherapy. Severe neutropenia, i.e., ANC of 0.5 × 10 9 A blood level below 1 / L can cause infections and organ failure in patients, which can be life-threatening.

[0237] In some embodiments, the neutropenia is Grade 1 neutropenia. In some embodiments, the neutropenia is Grade 2 neutropenia. In some embodiments, the neutropenia is Grade 3 neutropenia. In some embodiments, the neutropenia is Grade 4 neutropenia or severe neutropenia. In some embodiments, the neutropenia is febrile neutropenia (FN).

[0238] Thus, in some embodiments, there is provided a method for treating or preventing neutropenia in an individual in need thereof (e.g., a human, e.g., a human with cancer), comprising administering to the individual an effective amount of any of the pharmaceutical compositions described herein (e.g., a G-CSF-Fc dimer pharmaceutical composition, e.g., an efvemarenograstim alfa pharmaceutical composition) after (e.g., at least 24 hours after) administration of a neutropenia-inducing agent, e.g., a neutropenia-inducing chemotherapeutic agent.

[0239] Diseases treated with chemotherapy In some embodiments, the individual is administered a chemotherapeutic agent to treat the cancer.

[0240] Cancers that may be treated include, but are not limited to, colorectal cancer, breast cancer, gastric cancer, prostate cancer, ovarian cancer, cervical cancer, melanoma, liver cancer, head and neck cancer, glioma, gallbladder cancer, pancreatic cancer, prostate cancer, ampulla of Vater cancer, esophageal cancer, renal cancer, thyroid cancer, squamous cell carcinoma, lung cancer, B-cell lymphoma, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), non-Hodgkin's lymphoma (NHL), Burkitt's lymphoma, Wilms' tumor, etc. In some embodiments, the cancer is a solid cancer. In some embodiments, the cancer is a liquid cancer.

[0241] In some embodiments, the cancer is a non-myeloid cancer, e.g., a non-myeloid malignancy. In some embodiments, the cancer is selected from the group consisting of breast cancer, non-small cell lung cancer, small cell lung cancer, ovarian cancer, sarcoma, urothelial carcinoma, germ cell tumors, and non-Hodgkin's lymphoma. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is stage I-III or stage II-IV breast cancer. In some embodiments, the breast cancer is metastatic breast cancer. In some embodiments, the breast cancer is non-metastatic breast cancer.

[0242] In some embodiments, the methods of administering a G-CSF dimer pharmaceutical composition described herein ensure that an individual does not delay their chemotherapy treatment schedule, or that the chemotherapy treatment schedule is delayed by no more than about 5 days, e.g., about 4.5, 4, 3.5, 3, 2.5, 2, 1.8, 1.6, 1.4, 1.2, 1, 0.8, 0.6, 0.4, 0.2, 0.1 days, or less. In some embodiments, when two or more cycles of chemotherapy are administered, the methods of administering a G-CSF dimer pharmaceutical composition described herein ensure that an individual does not delay their chemotherapy treatment schedule within each cycle, or that the delay is only in cycle 2 (e.g., no more than about 5 days), or that the delay is not in all cycles (e.g., only in cycles 2 and 3 of four cycles).

[0243] chemotherapy drugs In some embodiments, the chemotherapeutic agent is a myelosuppressive chemotherapeutic agent selected from the group consisting of epirubicin, docetaxel, cyclophosphamide, doxorubicin, etoposide, cisplatin, paclitaxel, topotecan, vincristine, methylprednisolone, cytarabine, and combinations thereof.

[0244] In some embodiments, the cancer being treated with the chemotherapeutic agent is breast cancer. In some embodiments, the individual is treated with i) epirubicin and cyclophosphamide (e.g., epirubicin 100 mg / m 2and cyclophosphamide 600 mg / m 2 ), ii) docetaxel and cyclophosphamide (e.g., docetaxel 75 mg / m 2 and cyclophosphamide 600 mg / m 2 ), iii) doxorubicin and cyclophosphamide, iv) docetaxel and doxorubicin (e.g., docetaxel 75 mg / m 2 and doxorubicin 60 mg / m 2 ), or v) two or more chemotherapeutic agents including docetaxel, doxorubicin, and cyclophosphamide. In some embodiments, the two or more chemotherapeutic agents are administered simultaneously (in the same formulation or in separate formulations). In some embodiments, the two or more chemotherapeutic agents are administered sequentially.

[0245] In some embodiments, the chemotherapeutic agent induces neutropenia (e.g., neutropenia of any grade), such as Grade 4 neutropenia. In some embodiments, the chemotherapeutic agent induces FN. In some embodiments, the chemotherapeutic agent reduces the ANC to about 0.5×10 9 / L. In some embodiments, the chemotherapeutic agent decreases the ANC, e.g., by at least about any of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%. In some embodiments, the chemotherapeutic agent induces a duration of neutropenia (DN) of at least about 2 days, e.g., at least about any of 3, 4, 5, 6, 7, 8, 9, 10 days, or more.

[0246] VII. SYSTEMS, KITS, AND PREPARATION METHODS Systems, kits, and / or methods for preparing the pharmaceutical compositions (eg, efvemarenograstim alfa pharmaceutical compositions) described herein are also provided.

[0247] In some embodiments, lyophilized pharmaceutical compositions are provided. Lyophilized formulations adapted for subcutaneous administration are described in WO 97 / 04801. Such lyophilized pharmaceutical compositions may be reconstituted with a suitable diluent to a high protein concentration, and the reconstituted formulation can be administered subcutaneously to an individual to be imaged, diagnosed, or treated herein.

[0248] Pharmaceutical compositions used for in vivo administration must be sterile. This is readily achieved, for example, by filtration through a sterile filtration membrane. In some embodiments, a syringe containing any of the pharmaceutical compositions described herein (e.g., a G-CSF-Fc dimer pharmaceutical composition, e.g., an efvemarenograstim alfa pharmaceutical composition) is also provided. In some embodiments, the syringe is sterile. In some embodiments, the syringe is for single use. In some embodiments, the syringe is pre-filled with about 1 mL of a pharmaceutical composition described herein.

[0249] Kits containing any one of the pharmaceutical compositions described herein (e.g., efvemarenograstim alfa pharmaceutical compositions) are also provided. The kits may be useful in any of the methods for treating or preventing a disease or condition described herein.

[0250] In some embodiments, a kit is provided that includes a G-CSF dimer (e.g., a G-CSF-Fc dimer, e.g., efvemarenograstim alfa). In some embodiments, the kit further includes other components necessary for preparing a G-CSF dimer pharmaceutical composition, such as any of the pharmaceutical compositions described herein.

[0251] In some embodiments, the kit further comprises a device capable of delivering the pharmaceutical composition into an individual. One type of device for applications such as parenteral administration is a syringe used to inject a pharmaceutical composition into the body of a subject.

[0252] In some embodiments, the kit further comprises one or more additional therapeutic agents for treating a disease or condition, e.g., cancer. In some embodiments, the therapeutic agent is a chemotherapeutic agent.

[0253] The kits of the present application can be placed in suitable packaging, including but not limited to vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), etc. The kits may optionally provide additional components, such as buffers and instructional information.

[0254] Accordingly, the present application also provides an article of manufacture. The article of manufacture may include a container and a label or package insert on or associated with the container. Suitable containers include vials (e.g., sealed vials), bottles, jars, flexible packaging, and the like. Generally, the container holds the composition and may have a sterile access port (e.g., the container may be an intravenous solution bag or vial with a stopper pierceable by a hypodermic needle). The label or package insert indicates that the composition is used for imaging, diagnosing, or treating a particular condition in an individual. The label or package insert may further include instructions for administering the composition to an individual and / or imaging the individual. The label may also indicate instructions for reconstitution and / or use. The container holding the pharmaceutical composition may be a multi-use vial that allows for repeated administration (e.g., 2-6 administrations) of the reconstituted formulation. Package insert refers to the instructions typically included in commercial packaging for diagnostic or therapeutic products, including information regarding directions, usage, dosage, administration, contraindications, and / or warnings for use of the diagnostic product. Additionally, the article of manufacture may further comprise a second container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0255] The kit or article of manufacture may include multiple unit doses of the composition and instructions for use packaged in an amount sufficient for storage and use in pharmacies, eg, hospital pharmacies and compounding pharmacies.

[0256] In some embodiments, methods of producing any of the pharmaceutical compositions described herein are provided, comprising one or more (e.g., all) of the following steps: (i) culturing host cells under conditions suitable for expression of a G-CSF dimer (e.g., any of the G-CSF dimers described herein, such as efvemarenograstim alfa); (ii) isolating the expressed G-CSF dimer from the cell culture; (iii) purifying the expressed G-CSF dimer; and (iv) formulating the purified G-CSF dimer with a buffer, stabilizer, tonicity agent, and surfactant. In some embodiments, the purification step comprises one or more of affinity chromatography, viral inactivation, ion exchange chromatography, mixed-mode chromatography, and filtration. In some embodiments, methods of producing any of the pharmaceutical compositions described herein are provided, comprising formulating the purified G-CSF dimer with a buffer, stabilizer, tonicity agent, and surfactant, such as those described herein. In some embodiments, the method further comprises adjusting the pH of the pharmaceutical composition. In some embodiments, the method further comprises filling the pharmaceutical composition into a syringe. See also Example 7 and Table 13 for exemplary methods.

[0257] The present application also provides isolated nucleic acids encoding any of the G-CSF dimers described herein, vectors and host cells comprising such isolated nucleic acids, and recombinant methods for producing the G-CSF dimers. In some embodiments, the nucleic acid encoding the G-CSF dimer comprises the nucleic acid sequence of SEQ ID NO: 11.

[0258] Those skilled in the art will recognize that several embodiments are possible within the scope and spirit of the present invention. The present invention will now be described in more detail by reference to the following non-limiting examples. The following examples further illustrate the present invention but, of course, should not be construed as in any way limiting its scope.

[0259] VIII. Illustrative Embodiments In some embodiments, a pharmaceutical composition is provided that comprises (or consists essentially of, or consists of) (a) about 1 mg / mL to about 100 mg / mL of G-CSF dimer, the G-CSF dimer comprising two monomeric subunits each comprising the amino acid sequence of SEQ ID NO: 1; (b) about 1 mM to about 50 mM of a buffer; (c) about 0.1 mM to about 20 mM of a stabilizer; (d) about 1% (w / v) to about 15% (w / v) (e.g., about 1% (w / v) to about 10% (w / v)) of a tonicity agent; and (e) about 0.001% (w / v) to about 0.1% (w / v) of a surfactant. In some embodiments, a pharmaceutical composition is provided that comprises (or consists essentially of, or consists of) (a) about 1 mg / mL to about 100 mg / mL of a G-CSF dimer, each of which comprises two monomer subunits, each of which comprises a G-CSF monomer (e.g., SEQ ID NO: 1), an Fc fragment (e.g., any of SEQ ID NOs: 2 to 5), and an optional linker (e.g., SEQ ID NO: 12) connecting the G-CSF monomer and the Fc fragment; (b) about 1 mM to about 50 mM of a buffer; (c) about 0.1 mM to about 20 mM of a stabilizer; (d) about 1% (w / v) to about 15% (w / v) (e.g., about 1% (w / v) to about 10% (w / v)) of a tonicity agent; and (e) about 0.001% (w / v) to about 0.1% (w / v) of a surfactant. In some embodiments, a pharmaceutical composition is provided comprising (or consisting essentially of, or consisting of) (a) about 1 mg / mL to about 100 mg / mL of G-CSF dimer comprising two monomeric subunits each comprising (or consisting essentially of, or consisting of) the amino acid sequence of SEQ ID NO: 6; (b) about 1 mM to about 50 mM of buffer; (c) about 0.1 mM to about 20 mM of stabilizer; (d) about 1% (w / v) to about 15% (w / v) (e.g., about 1% (w / v) to about 10% (w / v)) of a tonicity agent; and (e) about 0.001% (w / v) to about 0.1% (w / v) of a surfactant.In some embodiments, a pharmaceutical composition is provided comprising (or consisting essentially of, or consisting of): (a) about 1 mg / mL to about 100 mg / mL of G-CSF dimer that is efvemarenograstim alfa; (b) about 1 mM to about 50 mM of a buffering agent; (c) about 0.1 mM to about 20 mM of a stabilizer; (d) about 1% (w / v) to about 15% (w / v) (e.g., about 1% (w / v) to about 10% (w / v)) of an isotonicity agent; and (e) about 0.001% (w / v) to about 0.1% (w / v) of a surfactant. In some embodiments, a pharmaceutical composition is provided comprising (or consisting essentially of, or consisting of) (a) about 1 mg / mL to about 100 mg / mL of G-CSF dimer comprising two monomeric subunits each comprising (or consisting essentially of, or consisting of) the amino acid sequence of SEQ ID NO: 7; (b) about 1 mM to about 50 mM of buffer; (c) about 0.1 mM to about 20 mM of stabilizer; (d) about 1% (w / v) to about 15% (w / v) (e.g., about 1% (w / v) to about 10% (w / v)) of a tonicity agent; and (e) about 0.001% (w / v) to about 0.1% (w / v) of a surfactant. In some embodiments, a pharmaceutical composition is provided comprising (or consisting essentially of, or consisting of) (a) about 1 mg / mL to about 100 mg / mL of G-CSF dimer comprising two monomeric subunits each comprising (or consisting essentially of, or consisting of) the amino acid sequence of SEQ ID NO: 8; (b) about 1 mM to about 50 mM of buffer; (c) about 0.1 mM to about 20 mM of stabilizer; (d) about 1% (w / v) to about 15% (w / v) (e.g., about 1% (w / v) to about 10% (w / v)) of a tonicity agent; and (e) about 0.001% (w / v) to about 0.1% (w / v) of a surfactant.In some embodiments, a pharmaceutical composition is provided comprising (or consisting essentially of, or consisting of) (a) about 1 mg / mL to about 100 mg / mL of G-CSF dimer comprising two monomeric subunits each comprising (or consisting essentially of, or consisting of) the amino acid sequence of SEQ ID NO: 9; (b) about 1 mM to about 50 mM of buffer; (c) about 0.1 mM to about 20 mM of stabilizer; (d) about 1% (w / v) to about 15% (w / v) (e.g., about 1% (w / v) to about 10% (w / v)) of an isotonic agent; and (e) about 0.001% (w / v) to about 0.1% (w / v) of a surfactant. In some embodiments, a pharmaceutical composition is provided comprising (or consisting essentially of, or consisting of) (a) about 1 mg / mL to about 100 mg / mL of G-CSF dimer comprising two monomeric subunits each comprising (or consisting essentially of, or consisting of) the amino acid sequence of SEQ ID NO: 10; (b) about 1 mM to about 50 mM of buffer; (c) about 0.1 mM to about 20 mM of stabilizer; (d) about 1% (w / v) to about 15% (w / v) (e.g., about 1% (w / v) to about 10% (w / v)) of a tonicity agent; and (e) about 0.001% (w / v) to about 0.1% (w / v) of a surfactant. In some embodiments, the concentration of the G-CSF dimer is about 5 mg / mL to about 50 mg / mL, e.g., about 10 mg / mL, about 15 mg / mL, about 20 mg / mL, about 25 mg / mL, or about 30 mg / mL. In some embodiments, the concentration of the G-CSF dimer is about 18 mg / mL to about 22 mg / mL, e.g., about 20 mg / mL. In some embodiments, the buffer is sodium acetate. In some embodiments, the concentration of sodium acetate is about 1 mg / mL to about 30 mg / mL, e.g., about 10 mg / mL. In some embodiments, the stabilizer is EDTA. In some embodiments, the concentration of EDTA is about 0.1 mM to about 10 mM, e.g., about 0.2 mM, about 0.5 mM, about 1 mM, about 1.5 mM, about 2 mM, or about 5 mM. In some embodiments, the concentration of EDTA is about 1 mM. In some embodiments, the concentration of EDTA is about 0.5 mM. In some embodiments, the tonicity agent is sorbitol.In some embodiments, the pharmaceutical composition comprises about 1% (w / v) to about 8% (w / v) sorbitol, e.g., about 1% (w / v), about 2% (w / v), 3% (w / v), about 4% (w / v), about 5% (w / v), about 6% (w / v), about 7% (w / v), or about 8% (w / v) sorbitol. In some embodiments, the pharmaceutical composition comprises about 5% (w / v) sorbitol. In some embodiments, the tonicity agent is sucrose. In some embodiments, the pharmaceutical composition comprises about 5% (w / v) to about 15% (w / v) sucrose, e.g., about 9% (w / v) sucrose. In some embodiments, the surfactant is selected from the group consisting of polysorbates, poloxamers, polyoxyethylene alkyl ethers, alkylphenyl polyoxyethylene ethers, and combinations thereof. In some embodiments, the surfactant is a non-ionic surfactant. In some embodiments, the surfactant is polysorbate 20 (PS20) or polysorbate 80 (PS80). In some embodiments, the pharmaceutical composition contains about 0.005% (w / v) to about 0.05% (w / v) PS20, e.g., about 0.006% (w / v), about 0.007% (w / v), 0.008% (w / v), about 0.009% (w / v), about 0.01% (w / v), about 0.015% (w / v), about 0.02% (w / v), or about 0.025% (w / v) PS20. In some embodiments, the pharmaceutical composition contains less than about 0.03% (w / v) PS20. In some embodiments, the pharmaceutical composition contains about 0.01% (w / v) PS20. In some embodiments, the pH of the pharmaceutical composition is about 4.2 to about 6.2, e.g., about 4.8 to about 5.8, about 5.0 to about 5.4, or about 5.2. In some embodiments, the pH of the pharmaceutical composition is about 5.2. In some embodiments, the pharmaceutical composition is stable at 25±2°C for at least about 1 month (e.g., 1, 2, or 3 months, or at least about 2 months). In some embodiments, the pharmaceutical composition is stable at 25±2°C for up to about 3 months.In some embodiments, the pharmaceutical composition has a purity of at least about 90% (e.g., at least about any of 91%, 92%, 93%, 94%, 95%, 99%, or more) as measured by SE-HPLC. In some embodiments, the pharmaceutical composition contains about 2% or less aggregates. In some embodiments, the pharmaceutical composition has an osmolality of about 310 mOsm / kg. In some embodiments, the pharmaceutical composition comprises at least about 96% G-CSF dimer as measured by SE-HPLC after storage at about 2°C to about 8°C for at least about 1 month (e.g., at least about 2 months, e.g., at least about any of 6 months, 12 months, 24 months, 36 months, or more). In some embodiments, the pharmaceutical composition comprises at least about 96% G-CSF dimer as measured by SE-HPLC after storage at about 2°C to about 8°C for up to 36 months. In some embodiments, the pharmaceutical composition comprises at least about 95.5% G-CSF dimer as measured by rCE-SDS after storage at about 2°C to about 8°C for at least about 1 month (e.g., at least about 2 months, e.g., at least about 6 months, 12 months, 24 months, 36 months, or more). In some embodiments, the pharmaceutical composition comprises at least about 95.5% G-CSF dimer as measured by rCE-SDS after storage at about 2°C to about 8°C for up to 36 months. In some embodiments, the pharmaceutical composition comprises at least about 94% G-CSF dimer as measured by rCE-SDS after storage at about 2°C to about 8°C for at least about 1 month (e.g., at least about 2 months, e.g., at least about 6 months, 12 months, 24 months, 36 months, or more). In some embodiments, the pharmaceutical composition comprises at least about 94% G-CSF dimer as measured by nrCE-SDS after storage...

Claims

1. (a) Granulocyte colony-stimulating factor (G-CSF) dimers in a concentration of approximately 1 mg / mL to approximately 100 mg / mL, (b) A buffering agent of approximately 1 mM to approximately 50 mM, (c) EDTA of approximately 0.1 mM to approximately 20 mM, (d) Approximately 1% (w / v) to approximately 15% (w / v) of sorbitol or sucrose, (e) Approximately 0.001% (w / v) to approximately 0.1% (w / v) of surfactant and Includes, A pharmaceutical composition having a pH of approximately 4.2 to approximately 6.

2.

2. The pharmaceutical composition according to claim 1, wherein the G-CSF dimer comprises two monomer subunits, each monomer subunit comprising a G-CSF monomer and a dimerizing domain.

3. (i) The G-CSF monomer comprises the amino acid sequence of SEQ ID NO: 1; (ii) The G-CSF monomer is connected to the dimerization domain via a linker within each monomer subunit; (iii) The dimerization domain comprises at least a portion of the Fc fragment; and / or (iv) The G-CSF monomer is located at the N-terminal side of the dimerization domain within each monomer subunit. The pharmaceutical composition according to claim 2.

4. The G-CSF monomer is connected to the dimerization domain via a linker within each monomer subunit, The linker, (i) The length is approximately 6 to 30 amino acids, and / or (ii) Containing the amino acid sequence of Sequence ID No. 12, The pharmaceutical composition according to claim 3.

5. The dimerized domain comprises at least a portion of the Fc fragment, The Fc fragment is (i) including CH2 and CH3 domains; (ii) Derived from IgG1 Fc, IgG2 Fc, IgG4 Fc, or fragments or variants thereof; and / or (iii) Containing an amino acid sequence selected from the group consisting of Sequence IDs 2 to 5, The pharmaceutical composition according to claim 3.

6. The pharmaceutical composition according to claim 2, wherein each monomeric subunit comprises the amino acid sequence of any of SEQ ID NOs: 6 to 10, or a variant thereof having at least about 90% sequence identity with the amino acid sequences of SEQ ID NOs: 6 to 10.

7. The pharmaceutical composition according to claim 6, wherein each monomer subunit contains the amino acid sequence of SEQ ID NO:

6.

8. The pharmaceutical composition according to claim 7, wherein the G-CSF dimer is fbemalenograstim alpha.

9. The pharmaceutical composition according to claim 1, wherein the concentration of the G-CSF dimer is about 5 mg / mL to about 50 mg / mL.

10. The pharmaceutical composition according to claim 9, wherein the concentration of the G-CSF dimer is about 20 mg / mL.

11. The pharmaceutical composition according to claim 1, wherein the buffering agent is sodium acetate.

12. The pharmaceutical composition according to claim 11, wherein the concentration of sodium acetate is about 1 mM to about 30 mM.

13. The pharmaceutical composition according to claim 12, wherein the concentration of the sodium acetate is about 10 mM.

14. The pharmaceutical composition according to claim 13, wherein the concentration of the EDTA is about 0.5 mM or about 1 mM.

15. The pharmaceutical composition according to claim 1, comprising approximately 1% (w / v) to approximately 8% (w / v) of sorbitol.

16. The pharmaceutical composition according to claim 15, comprising approximately 5% (w / v) sorbitol.

17. The pharmaceutical composition according to claim 1, comprising approximately 5% (w / v) to approximately 15% (w / v) sucrose.

18. The pharmaceutical composition according to claim 17, comprising approximately 9% (w / v) sucrose.

19. The pharmaceutical composition according to claim 1, wherein the surfactant is selected from the group consisting of polysorbate, poloxamer, polyoxyethylene alkyl ether, alkylphenyl polyoxyethylene ether, and combinations thereof.

20. The pharmaceutical composition according to claim 19, wherein the surfactant is polysorbate 20 (PS20) or polysorbate 80 (PS80).

21. The pharmaceutical composition according to claim 20, wherein the surfactant is PS20, and the pharmaceutical composition contains about 0.005% (w / v) to about 0.05% (w / v) of PS20.

22. The pharmaceutical composition according to claim 21, comprising approximately 0.01% (w / v) of PS20.

23. The pharmaceutical composition according to claim 1, wherein the pH is approximately 5.0 to approximately 5.

4.

24. The pharmaceutical composition according to claim 23, wherein the pH is approximately 5.

2.

25. (a) G-CSF dimer at approximately 20 mg / mL, (b) Approximately 10 mM sodium acetate and (c) EDTA of approximately 1 mM, (d) Approximately 5% (w / v) sorbitol and (e) containing approximately 0.01% (w / v) PS20, The pharmaceutical composition according to claim 1, wherein the pH is approximately 5.

2.

26. The pharmaceutical composition according to claim 1, contained in a syringe.

27. ​​(i) The volume of the pharmaceutical composition in the syringe is about 1 mL; (ii) The syringe is for single use only; and / or (iii) The syringe is sterile. The pharmaceutical composition according to claim 26.

28. (i) The pharmaceutical composition has a purity of at least about 90% of the G-CSF dimer when evaluated by size exclusion high-performance liquid chromatography (SE-HPLC); (ii) The pharmaceutical composition contains less than 2.5% of the total peak area when measured by SE-HPLC after being stored at 25±2°C for at least about one month; (iii) When the pharmaceutical composition is stored at approximately 2°C to approximately 8°C for 36 months and then measured by SE-HPLC, it contains at least approximately 85% of the total area of ​​all peaks; (iv) The pharmaceutical composition contains at least 95.5% G-CSF dimers when evaluated by reduced capillary electrophoresis-sodium dodecyl sulfate (rCE-SDS) after being stored at approximately 2°C to approximately 8°C for 36 months; (v) The pharmaceutical composition contains approximately 1.5% or less of high molecular weight species (HMWS) when measured by rCE-SDS after being stored at 25±2°C for at least about one month; (vi) The pharmaceutical composition contains about 1.5% or less HMWS when measured by rCE-SDS after being stored at about 2°C to about 8°C for at least about 36 months; (vii) The pharmaceutical composition has a shelf life of at least 36 months; (viiii) The pharmaceutical composition is stable for 36 months at approximately 2°C to approximately 8°C; (ix) The pharmaceutical composition has a total protein aggregation rate of less than about 5%; (x) The pharmaceutical composition has at least about 70% of its potency after being stored at 25±2°C for at least about one month; or (xi) The pharmaceutical composition, after being stored at approximately 2°C to approximately 8°C for at least approximately 36 months, has at least approximately 70% potency. A pharmaceutical composition according to any one of claims 1 to 27.

29. A pharmaceutical composition for use in the treatment or prevention of a disease or condition in an individual, according to any one of claims 1 to 27, wherein the use comprises administering an effective amount of the pharmaceutical composition to the individual.

30. The pharmaceutical composition for use according to claim 29, wherein the disease or condition is selected from the group consisting of neutropenia, stroke, spinal cord injury, neurological disorders involving damage to the blood-brain barrier, Parkinson's disease, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, spinal muscular atrophy, primary lateral sclerosis, spinocerebellar ataxia, and mobilization of hematopoietic stem cells into the peripheral blood in allogeneic hematopoietic stem cell transplantation.

31. The pharmaceutical composition for use according to claim 30, wherein the disease or condition is neutropenia.

32. The pharmaceutical composition for use according to claim 31, wherein the neutropenia is chemotherapy-induced neutropenia or radiotherapy-induced neutropenia.

33. The pharmaceutical composition for use according to claim 32, wherein the use comprises administering an effective amount of the pharmaceutical composition to the individual after administering a chemotherapeutic agent to the individual.

34. The pharmaceutical composition for use according to claim 33, wherein the chemotherapeutic agent is a myelosuppressive chemotherapeutic agent.

35. The pharmaceutical composition for use according to claim 33, wherein the use comprises administering the chemotherapeutic agent to the individual for at least four cycles, and administering the pharmaceutical composition to the individual after the administration of the chemotherapeutic agent in each of the at least four cycles.

36. (i) The pharmaceutical composition is for administering about 5 mg to about 25 mg of the G-CSF dimer per dose; (ii) The pharmaceutical composition is to be administered to the individual at least 24 hours after the administration of the chemotherapeutic agent in each of the four cycles; (iii) The pharmaceutical composition is intended for subcutaneous administration; and / or (iv) The individual is a human, A pharmaceutical composition for use according to claim 35.

37. In an individual, (i) Neutrophil progenitor cells, (ii) Myeloid stem cells, and (iii) Mature neutrophils A pharmaceutical composition according to any one of claims 1 to 27 for use in one or more activations, A pharmaceutical composition for use, wherein the use comprises administering an effective amount of the pharmaceutical composition to the individual.

38. A pharmaceutical composition according to any one of claims 1 to 27 for use in the treatment of cancer in an individual, A pharmaceutical composition for use, wherein the use comprises administering an effective amount of a chemotherapeutic agent and an effective amount of the pharmaceutical composition to the individual.

39. (i) Culturing host cells under conditions suitable for expressing the G-CSF dimer, (ii) Isolating the expressed G-CSF dimer from the cell culture, (iii) Purify the expressed G-CSF dimer, (iv) A method for producing the pharmaceutical composition according to any one of claims 1 to 27, comprising formulating the purified G-CSF dimer together with the buffer, the EDTA, the sorbitol or sucrose, and the surfactant.

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