Formulations comprising G-CSF and uses thereof

Abstract

Described herein are compositions of a G-CSF dimer, which comprise a G-CSF-Fc dimer. Also provided are methods, systems, kits and methods of preparation for treating or preventing a disease or condition with the compositions.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This patent application claims priority to PCT / CN2022 / 113560 filed on August 19, 2022, the entire contents of which are incorporated herein by reference.

[0003] Reference Electronic Sequence Listing

[0004] The entire contents of the electronic sequence listing (720622001941SEQLIST.xml; size: 34,294 bytes; creation date: August 10, 2023) are incorporated herein by reference. Technical Field

[0005] The present disclosure relates in some aspects to formulations (eg, pharmaceutical compositions) of granulocyte colony stimulating factor (G-CSF) and uses thereof. Background Art

[0006] The present embodiments generally relate to G-CSF formulations that can be used for injection and general administration.

[0007] G-CSF is a blood-forming protein 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 growth and differentiation of various types of blood cells, such as neutrophils, which play a key role in the body's defense against bacterial infections. G-CSF can also activate mature neutrophils to participate in immune responses, or work in synergy with other hematopoietic growth factors such as stem cell factor, Flt-3 ligand, and GM-CSF to perform hematopoietic functions.

[0008] G-CSF formulations can be administered to patients by subcutaneous injection. Both liquid and lyophilized formulations can be used for pharmaceuticals. However, high-concentration G-CSF protein formulations may present many challenges in formulation development, especially for liquid formulations. At high protein concentrations, the stability of the protein may be problematic due to increased protein-protein interactions, resulting in increased formation of soluble and insoluble protein aggregates. If the protein concentration approaches its apparent solubility limit, phase separation may occur through precipitation, gelation and / or crystallization. In addition, for formulations intended for intramuscular or subcutaneous administration and for the convenience of healthcare providers and patients, a proportionally high concentration of stabilizer may be required to achieve long-term protein stability. The resulting formulation may be hypertonic, which may cause injection pain due to tissue damage. Therefore, balancing the amount of stabilizer is critical for the stability and osmolality of concentrated protein formulations.

[0009] For these reasons, there is a need to develop G-CSF formulations with high protein concentrations without significantly increasing protein aggregation, osmolarity, and / or decreasing protein stability. Summary of the invention

[0010] In some aspects, compositions of G-CSF are disclosed herein. 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 a G-CSF dimer; (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) of a tonicity agent; and (e) about 0.001% (w / v) to about 0.1% (w / v) of a surfactant.

[0011] In some embodiments according to any of the above pharmaceutical compositions, the G-CSF dimer comprises two monomer subunits, wherein each monomer subunit 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 by an optional linker (e.g., within each monomer subunit). In some embodiments, the length of the linker is about 6 to about 30 amino acids. 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 CH2 and CH3 domains. 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 SEQ ID NO: 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 one 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 one of SEQ ID NOs: 6-10. In some embodiments, each monomer subunit comprises an amino acid sequence selected from SEQ ID NOs: 6-10 (e.g., SEQ ID NO: 6).

[0012] In some embodiments according to any of the pharmaceutical compositions above, the G-CSF dimer is efbemalenograstim alfa.

[0013] In some embodiments according to any of the pharmaceutical compositions above, the concentration of the G-CSF dimer is about 5 mg / mL to about 50 mg / mL, such as about 20 mg / mL.

[0014] In some embodiments according to any of the pharmaceutical compositions above, the buffer is sodium acetate. In some embodiments, the concentration of sodium acetate is about 1 mM to about 30 mM, such as about 10 mM.

[0015] In some embodiments according to any of the pharmaceutical compositions above, the stabilizer is EDTA. In some embodiments, the concentration of EDTA is about 0.1 mM to about 10 mM, such as about 1 mM.

[0016] In some embodiments according to any of the above-mentioned pharmaceutical compositions, 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) of sorbitol, for example, about 5% (w / v) of 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) of sucrose, for example, about 9% (w / v) of sucrose.

[0017] In some embodiments according to any of the above-mentioned pharmaceutical compositions, the surfactant is selected from 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) of PS20, for example, about 0.01% (w / v) of PS20.

[0018] In some embodiments according to any of the above pharmaceutical compositions, the pH of the pharmaceutical composition is about 4.2 to about 6.2, such as about 4.8 to about 5.8, about 5.0 to about 5.4, or about 5.2.

[0019] In some embodiments according to any of the above pharmaceutical compositions, the pharmaceutical composition comprises (or consists essentially of, or consists of): (a) about 20 mg / mL of 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.

[0020] In some embodiments according to any of the above pharmaceutical compositions, the pharmaceutical composition is stable at 25±2° C. for at least about 1 month, such as at 25±2° C. for at least about 2 months. In some embodiments, the pharmaceutical composition is stable at 25±2° C. for up to about 3 months.

[0021] In some embodiments of the pharmaceutical composition according to any of the above, the purity of the G-CSF dimer of the pharmaceutical composition is at least about 90% as assessed by size exclusion-high performance liquid chromatography (SE-HPLC).

[0022] In some embodiments according to any of the pharmaceutical compositions above, the osmolarity of the pharmaceutical composition is about 268 mOsm / kg to about 360 mOsm / kg.

[0023] In some embodiments according to any of the above pharmaceutical compositions, the pharmaceutical composition comprises a main peak of at least about 85% 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 (e.g., at least about 2 months). In some embodiments, the pharmaceutical composition comprises a main peak of at least about 85% relative to the total area of ​​all peaks when measured by SE-HPLC after storage at 25±2°C for up to about 3 months.

[0024] In some embodiments according to any of the above pharmaceutical compositions, the pharmaceutical composition comprises less than about 2.5% 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 (e.g., at least about 2 months). In some embodiments, the pharmaceutical composition comprises less than about 2.5% dimer relative to the total area of ​​all peaks when measured by SE-HPLC after storage at 25±2°C for up to about 3 months.

[0025] In some embodiments according to any of the above pharmaceutical compositions, the pharmaceutical composition comprises no more than about 2% 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 (e.g., at least about 2 months). In some embodiments, the pharmaceutical composition comprises no more than about 2% aggregates relative to the total area of ​​all peaks when measured by SE-HPLC after storage at 25±2°C for up to about 3 months.

[0026] In some embodiments according to any of the above pharmaceutical compositions, 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 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.

[0027] In some embodiments of the pharmaceutical composition according to any of the above, the pharmaceutical composition comprises at least about 96% of G-CSF dimer as measured by reduced capillary electrophoresis sodium dodecyl sulfate (rCE-SDS) after storage at 25±2°C for at least about 1 month (e.g., at least about 2 months). In some embodiments, the pharmaceutical composition comprises at least about 96% of G-CSF dimer as measured by rCE-SDS after storage at 25±2°C for up to about 3 months.

[0028] In some embodiments of the pharmaceutical composition according to any of the above, 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 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 up to about 36 months.

[0029] In some embodiments according to any of the above pharmaceutical compositions, the pharmaceutical composition comprises no more than about 1.5% high molecular weight substances (HMWS) as measured by rCE-SDS after storage at 25±2°C for at least about 1 month (e.g., at least about 2 months). In some embodiments, the pharmaceutical composition comprises no more than about 1.5% HMWS as measured by rCE-SDS after storage at 25±2°C for up to about 3 months.

[0030] In some embodiments of the pharmaceutical composition according to any of the above, the pharmaceutical composition comprises no more than about 1.5% high molecular weight species as measured by rCE-SDS after storage for at least about 36 months at about 2° C. to about 8° C. In some embodiments, the pharmaceutical composition comprises no more than about 1.5% HMWS as measured by rCE-SDS after storage for up to about 36 months at about 2° C. to about 8° C.

[0031] In some embodiments of the pharmaceutical composition according to any of the 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 at 25±2°C for at least about 1 month (e.g., at least about 2 months). In some embodiments, the pharmaceutical composition comprises at least about 94% G-CSF dimer as measured by nrCE-SDS after storage at 25±2°C for up to about 3 months.

[0032] In some embodiments of the pharmaceutical composition according to any of the above, the pharmaceutical composition comprises 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 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.

[0033] In some embodiments according to any of the above pharmaceutical compositions, the pharmaceutical composition comprises no more than about 1.5% high molecular weight species as measured by nrCE-SDS after storage at 25±2°C for at least about 1 month (e.g., at least about 2 months). In some embodiments, the pharmaceutical composition comprises no more than about 1.5% HMWS as measured by nrCE-SDS after storage at 25±2°C for up to about 3 months.

[0034] In some embodiments of the pharmaceutical composition according to any of the above, the pharmaceutical composition comprises no more than about 1.5% of high molecular weight substances as measured by nrCE-SDS after storage at about 2°C to about 8°C for at least about 36 months. In some embodiments, the pharmaceutical composition comprises no more than about 1.5% of high molecular weight substances as measured by nrCE-SDS after storage at about 2°C to about 8°C for up to about 36 months.

[0035] In some embodiments according to any of the above pharmaceutical compositions, after storage at 25±2°C for at least about 1 month (e.g., at least about 2 months), the pharmaceutical composition comprises about 27.2% to about 39.0% of the acidic region, about 36.5% to about 60.1% of the main peak, and about 7.2% to about 30% of the basic region as measured by imaging capillary isoelectric focusing (icIEF). In some embodiments, after storage at 25±2°C for up to about 3 months, the pharmaceutical composition comprises about 27.2% to about 39.0% of the acidic region, about 36.5% to about 60.1% of the main peak, and about 7.2% to about 30% of the basic region as measured by icIEF.

[0036] In some embodiments according to any of the above pharmaceutical compositions, the pharmaceutical composition comprises about 27.2% to about 39.0% acidic region, about 36.5% to about 60.1% main peak, and about 7.2% to about 30% basic region as 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 comprises about 27.2% to about 39.0% acidic region, about 36.5% to about 60.1% main peak, and about 7.2% to about 30% basic region as measured by icIEF after storage at about 2°C to about 8°C for up to about 36 months.

[0037] According to some embodiments of any of the above pharmaceutical compositions, the pharmaceutical composition retains at least about 70% potency after storage at 25±2°C for at least about 1 month (e.g., at least about 2 months). In some embodiments, the pharmaceutical composition retains at least about 70% potency after storage at 25±2°C for up to about 3 months.

[0038] In some embodiments of the pharmaceutical composition according to any of the above, the pharmaceutical composition retains at least about 70% potency after storage for at least about 36 months at about 2°C to about 8°C. In some embodiments, the pharmaceutical composition retains at least about 70% potency after storage for up to about 36 months at about 2°C to about 8°C.

[0039] In some embodiments according to any of the above pharmaceutical compositions, the concentration of the pharmaceutical composition changes by less than about 0.5 mg / mL after storage at 25±2°C for at least about 1 month (e.g., at least about 2 months). In some embodiments, the concentration of the pharmaceutical composition changes by less than about 0.5 mg / mL after storage at 25±2°C for up to about 3 months.

[0040] In some embodiments of the pharmaceutical composition according to any of the above, the concentration of the pharmaceutical composition changes by less than about 0.5 mg / mL after storage for at least about 36 months 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 storage for up to about 36 months at about 2° C. to about 8° C.

[0041] In some embodiments according to any of the above pharmaceutical compositions, 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 disposable. In some embodiments, the syringe is sterile.

[0042] On the other hand, a syringe is provided, comprising any of the above-mentioned pharmaceutical compositions. In some embodiments, the syringe is disposable. In some embodiments, the volume of the liquid pharmaceutical composition in the syringe is about 1 mL. In some embodiments, the syringe is sterile.

[0043] On the other hand, a method for treating or preventing a disease or condition of an individual (e.g., human) is provided, comprising administering to an individual an effective amount of any pharmaceutical composition (e.g., liquid pharmaceutical composition) provided herein. In some embodiments, the pharmaceutical composition is administered at a dosage 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 neutropenia, stroke, spinal cord injury, a nervous system disease 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 to peripheral blood in allogeneic hematopoietic stem cell transplantation. In some embodiments, the disease or condition is neutropenia. In some embodiments, neutropenia is chemotherapy-induced neutropenia or radiotherapy-induced neutropenia. In some embodiments, the method comprises administering to the individual an effective amount of a pharmaceutical composition after administering to the individual a chemotherapeutic agent. In some embodiments, the chemotherapeutic agent is a myelosuppressive chemotherapeutic agent. In some embodiments, the pharmaceutical composition is administered once per chemotherapy cycle at a dose of about 20 mg. In some embodiments, the individual is administered at least 4 cycles of the chemotherapeutic agent, and wherein the pharmaceutical composition is administered after the chemotherapeutic agent is administered in each cycle of at least 4 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 for each administration.

[0044] In another aspect, a method of preparing any pharmaceutical composition disclosed herein is provided, comprising: (i) culturing host cells under conditions suitable for expressing 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 DESCRIPTION OF THE DRAWINGS

[0045] Figure 1is a diagram of the structure of an exemplary G-CSF dimer (e.g., abegramstim α). In the figure, "-" represents a linker, an oval object labeled "G-CSF" represents a G-CSF monomer, and an oval object labeled "Fc" represents an Fc fragment (e.g., a human IgG-derived Fc). An exemplary G-CSF dimer comprises two monomer subunits. Each monomer subunit from N' to C' comprises a G-CSF monomer, a linker, and an Fc fragment.

[0046] Figure 2 The figure is an exemplary chromatogram of reverse 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 storage at 25±2°C for 12 weeks. There are three main elution peaks, namely, impurity peak 1, impurity peak 2 and main peak.

[0047] Figure 3A-3B The G-CSF dimer pharmaceutical composition was stored at 25±2°C for up to 12 weeks by non-reducing SDS-PAGE ( Figure 3A ) and reducing SDS-PAGE ( Figure 3B ) measured by RT-PCR, the pharmaceutical composition comprising: (●) 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) sorbitol, 1 mM EDTA, 0.01% (w / v) polysorbate 20, pH 4.8, or (×) 10 mM sodium acetate, 9% (w / v) sorbitol, 1 mM EDTA, 0.01% (w / v) polysorbate 20, pH 5.2.

[0048] Figure 3C-3D The change in % main peak over time of the G-CSF dimer pharmaceutical composition measured by RP-HPLC after storage at 25±2°C for up to 12 weeks is shown ( Figure 3C ) and the change in % impurity peak 2 over time as measured by RP-HPLC ( Figure 3D), the pharmaceutical composition comprises: (●) 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) sorbitol, 1 mM EDTA, 0.01% (w / v) polysorbate 20, pH 4.8, or (×) 10 mM sodium acetate, 9% (w / v) sorbitol, 1 mM EDTA, 0.01% (w / v) polysorbate 20, pH 5.2.

[0049] Figure 4A-4B The G-CSF dimer pharmaceutical composition was stored at 25±2°C for up to 12 weeks by non-reducing SDS-PAGE ( Figure 4A ) and reducing SDS-PAGE ( Figure 4B ) measured by RT-PCR, the pharmaceutical composition comprising: (●) 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.

[0050] Figure 4C-4D The change in % main peak over time of the G-CSF dimer pharmaceutical composition measured by RP-HPLC after storage at 25±2°C for up to 12 weeks is shown ( Figure 4C ) and the change in % impurity peak 2 over time as measured by RP-HPLC ( Figure 4D ), the pharmaceutical composition comprises: (●) 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.

[0051] Figures 4E-4F The G-CSF dimer pharmaceutical composition was stored at 25±2°C for up to 12 weeks and analyzed by non-reducing SDS-PAGE ( Figure 4E) and reducing SDS-PAGE ( Figure 4F ) measured the change in % main band of G-CSF dimer, the pharmaceutical composition comprising: (×) 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.

[0052] Figure 4G-4H The change in % main peak over time of the G-CSF dimer pharmaceutical composition measured by RP-HPLC after storage at 25±2°C for up to 12 weeks is shown ( Figure 4G ) and the change in % impurity peak 2 over time as measured by RP-HPLC ( Figure 4H ), the pharmaceutical composition comprises: (×) 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.

[0053] Figure 5 The figure is a size exclusion high performance liquid chromatography (SE-HPLC) chromatogram of a G-CSF-Fc dimer pharmaceutical composition at week 0, the 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. There are two main elution peaks, namely the dimer and the main peak.

[0054] Figure 6Shown is the change in % dimer over time as measured by SE-HPLC of 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

[0055] The present application provides a liquid or lyophilized form of G-CSF formulation (e.g., a pharmaceutical composition) that is stable under suitable storage conditions. The present application is based on the surprising discovery that a formulation comprising a suitable excipient and a G-CSF molecule, in particular a G-CSF dimer (e.g., a G-CSF-Fc dimer, such as abegrastim α), is highly stable during storage. In some embodiments, the G-CSF formulation comprises at least about 97% (e.g., at least about 98%, 99%, or more) of G-CSF dimer as measured by size exclusion chromatography (SEC) before and after storage. In some embodiments, the G-CSF formulation comprises at least about 95.5% of G-CSF dimer as measured by rCE-SDS before and after storage (e.g., after storage at 25±2°C for at least about 1 month, or after storage at about 2°C to about 8°C for at least about 36 months). In some embodiments, the G-CSF formulation comprises at least about 94% (e.g., at least about 95%) of G-CSF dimer as measured by nrCE-SDS before and after storage (e.g., after at least about 1 month at 25±2°C, or after at least about 36 months at about 2°C to about 8°C). In some embodiments, the G-CSF formulation comprises no more than about 1.5% (e.g., no more than about 1%) of high molecular weight species (HMWS) as measured by rCE SDS before and after storage (e.g., after at least about 1 month at 25±2°C, or after at least about 36 months at about 2°C to about 8°C). In some embodiments, the G-CSF formulation comprises no more than about 1.5% (e.g., no more than about 0.5%) of HMWS as measured by nrCE-SDS before and after storage (e.g., after at least about 1 month at 25±2°C, or after at least about 36 months 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 after storage (e.g., after storage at 25±2°C for at least about 1 month, or after storage at about 2°C to about 8°C for at least about 36 months). In some embodiments, the G-CSF formulation retains at least about 70% (e.g., at least about 80%, 85%, 90%, 95%, 99% or more) of potency before and after storage (e.g., after storage at 25±2°C for at least about 1 month, or after storage at about 2°C to about 8°C for at least about 36 months). In some embodiments, the concentration of the G-CSF formulation varies by less than about 0.5 mg / mL before and after storage (e.g., after storage at 25±2°C for at least about 1 month, or after storage at about 2°C to about 8°C for at least about 36 months). These properties make the G-CSF formulation particularly suitable as a drug product that can be safely administered to human patients (e.g., by subcutaneous administration). In some embodiments, the pharmaceutical compositions described herein provide a high concentration of G-CSF (eg, G-CSF dimer) in a small volume (eg, 1 mL), which facilitates therapeutic administration or delivery.

[0056] In addition, the G-CSF formulations described herein (such as G-CSF-Fc dimer formulations) maintain the osmotic pressure molarity of the formulation to improve in vivo tolerance, thereby improving patient compliance. In some embodiments, the osmotic pressure molarity of the G-CSF formulation is about 268mOsm / kg to about 360mOsm / kg, for example, about 310mOsm / kg. In addition, the formulations described herein allow for convenient processing, including but not limited to ultrafiltration and sterile filtration, and injection of drug solutions through needles.

[0057] Thus, the present application provides, in one aspect, a pharmaceutical composition comprising (or consisting essentially of, or consisting of): (a) about 1 mg / mL to about 100 mg / mL of a G-CSF dimer (e.g., a G-CSF-Fc dimer, such as abegrastim α); (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, 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, a method of evaluating the suitability of a pharmaceutical composition for medical use in an individual (e.g., a human individual) is provided, wherein 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 dimer (e.g., a G-CSF-Fc dimer, such as abegrastim α); (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., (e) 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, wherein the pH of the pharmaceutical composition is about 4.2 to about 6.2 (e.g., about 5.0 to about 5.4, such as about 5.2), the method comprising 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 the 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 a combination of any one or more parameters of the pharmaceutical composition described herein, such as assessed by the corresponding method described herein.

[0058] Also provided are syringes, such as sterile and / or single-use syringes, containing any of the pharmaceutical compositions described herein (eg, albigstrom alpha pharmaceutical compositions).

[0059] 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.

[0060] All publications cited in this application (including patent documents, scientific articles and databases) are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated herein. If definitions set forth herein are contrary to or inconsistent with the meanings set forth in patents, applications, published applications and other publications incorporated herein by reference, the definitions set forth herein take precedence over the definitions incorporated herein by reference.

[0061] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0062] I. Definitions

[0063] As used herein, the term "viscosity" refers to the resistance to flow of a liquid formulation, such as the resistance to flow of a liquid formulation when injected through an injection needle during administration to a patient. Viscosity measurements can be performed by cone-plate technology, wherein a Peltier element is set at a specified temperature, such as 5°C to 25°C as described herein. Typically, a clear 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 mPa-S at 5°C to 25°C, where 1mPa-S=1cP. The high concentration, low viscosity, substantially isotonic formulations disclosed herein are typically characterized by a viscosity range of 1 to 35mPa-S at 5°C to 25°C.

[0064] As used herein, the term "osmotic pressure molar concentration" refers to the measurement of solute concentration, which is defined as the millimole of solute (non-ionized and ionized form) per kilogram of solution. The desired osmotic pressure molar concentration level can be reached by adding one or more stabilizers, such as sugar or sugar alcohol, including but not limited to mannitol, glucose (dextrose), glucose (glucose), trehalose and / or sucrose. Other stabilizers suitable for providing osmotic pressure molar concentration are described in the references, such as handbook of Pharmaceutical Excipients (Fourth Edition, Royal Pharmaceutical Society of Great Britain, Science & Practice Publishers) (" Pharmaceutical Excipients Handbook " (Fourth Edition, Royal Pharmaceutical Society of Great Britain, Science & Practice Publishers)) or Remingtons: The Science and Practice of Pharmacy (Nineteenth Edition, Mack Publishing Company) (" Remington: Pharmaceutical Science and Practice " (Nineteenth Edition, Mack Publishing Company)).

[0065] As used herein, the term "about" refers to + / -10% of the unit value provided. "About" values ​​or parameters mentioned herein include (and describe) variations for the value or parameter itself. For example, a description referring to "about X" includes a description of "X".

[0066] As used herein, the term "about XY" has the same meaning as "about X to about Y."

[0067] As used herein, the term "substantially" refers to the qualitative condition of exhibiting all or an approximate degree of a characteristic or property of interest. One of ordinary skill in the biological field will appreciate that biological and chemical phenomena rarely, if ever, achieve or avoid absolute results. Therefore, the term "substantially" is used herein to describe the potential imperfections inherent in many biological and chemical phenomena.

[0068] As used herein, the terms "isosmotic" and "isotonic" are used interchangeably with the terms "substantially isotonic" and "substantially isotonic," and refer to a formulation characterized by an osmotic pressure that is the same or at least substantially equal to the osmotic pressure of another solution, which is achieved by the formulation having a total concentration of solutes (including osmotic and non-osmotic solutes) that is the same or at least substantially equal to the total number of solutes in the other solution. Thus, while one of skill in the art will appreciate that "isotonic" and "iso-osmotic" formulations for in vivo administration typically have an osmotic pressure molarity 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 "isotonic," "isotonic," "substantially isotonic," and "substantially isotonic" are used interchangeably to refer to formulations having an osmotic pressure molarity of about 240 mOsm / kg to about 400 mOsm / kg, or about 270 mOsm / kg / kg to about 370 mOsm / kilogram, or about 300 mOsm / kg to about 330 mOsm / kg.

[0069] As used herein, the term "tonicity agent" or "tonicity excipient" refers to a compound intended to reduce local irritation caused by a pharmaceutical formulation / composition by preventing osmotic shock at the site of application. Typically added to injectable, ocular or nasal formulations, these excipients include, but are not limited to, potassium chloride, sodium chloride, mannitol, sorbitol, glucose and glycerol.

[0070] As used herein, the term "buffer" refers to a substance that helps maintain 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 certain aspects, a mixture of two or more buffers is used.

[0071] As used herein, the term "stabilizer" refers to a substance that helps maintain the desired properties of a product. Exemplary stabilizers include, but are not limited to, amino acids, mannitol, sorbitol, sucrose, trehalose, dextran 40, and any combination thereof.

[0072] As used herein, the term "salt" refers to inorganic salts, including but not limited to sodium chloride (NaCl), sodium sulfate (Na2SO4), sodium thiocyanate (NaSCN), magnesium chloride (MgCl2), magnesium sulfate (MgSO4), ammonium thiocyanate (NH4SCN), ammonium sulfate ((NH4)2SO4), ammonium chloride (NH4Cl), calcium chloride (CaCl2), sulfate (CaSO4) and zinc chloride (ZnCl2), etc., or combinations thereof.

[0073] As used herein, the term "surfactant" includes nonionic 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 to provide a stable, high concentration formulation of G-CSF dimer and other proteins is generally in the range of 0 ppm to 2000 ppm. The use of nonionic surfactants allows the formulation to be exposed to shear and surface stress without causing denaturation of G-CSF dimer or other proteins, and also reduces adsorption on surfaces during processing and storage. The formulations disclosed herein include but are not limited to formulations with one or more nonionic surfactants, including, for example, one or more polysorbates, such as polysorbate 20 or 80; one or more poloxamers, such as poloxamer 184 or 188. Examples of formulations containing polysorbates (such as polysorbate 20 ( 20) or polysorbate 80 ( 80))'s preparation.

[0074] As used herein, the term "protein" refers to an amino acid polymer containing at least five constituent amino acids covalently linked by peptide bonds. The constituent amino acids can be from amino acids encoded by the genetic code, which include: alanine, valine, leucine, isoleucine, methionine, phenylalanine, tyrosine, tryptophan, serine, threonine, asparagine, glutamine, cysteine, glycine, proline, arginine, histidine, lysine, aspartic acid and glutamic acid.

[0075] As used herein, the term "protein" is synonymous with the related terms "peptide" and "polypeptide."

[0076] As used herein, the term "antibody" includes full-length antibodies, bispecific antibodies, single-chain Fv (scFv) fragments, Fab fragments, Fab' fragments, F(ab')2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, V H H, Fv-Fc fusion, scFv-Fc fusion, scFv-Fv fusion, diabody, triabody and tetrabody. "Antibody" may refer to a class of proteins commonly known as immunoglobulins. Antibodies include full-length monoclonal antibodies (mAbs), such as IgG4 monoclonal antibodies, which include an immunoglobulin Fc region.

[0077] As used herein, the term "immunoglobulin" refers to a class of structurally related glycoproteins consisting of two pairs of polypeptide chains (a pair of light (L) low molecular weight chains and a pair of heavy (H) chains), all four of which are interconnected by disulfide bonds. The structure of immunoglobulins has been well characterized. For example, see Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, NY (1989)). In short, each heavy chain generally comprises a heavy chain variable region (abbreviated herein as V H ) and the heavy chain constant region (abbreviated herein as C H The heavy chain constant region usually contains three domains, C H 1. C H 2 and C H 3. Each light chain generally comprises a light chain variable region (abbreviated herein as V L ) and a light chain constant region. The light chain constant region generally comprises a domain, abbreviated herein as C L . V H and V L The V domains can be further subdivided into regions of hypervariability (or hypervariable regions that may be in the form of loops defined in sequence and / or structure), also called complementarity determining regions (CDRs), interspersed with regions that are more conserved, called framework regions (FRs). H and V L It usually contains three CDRs and four FRs, arranged from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and Lesk J. Mol. Biol. 196, 901-917 (1987)).

[0078] Unless otherwise specified herein, the numbering of amino acid residues in the IgG Fc region is according to the EU numbering system for antibodies, also known as the EU index, as described by Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0079] The "CH1 domain" (also referred to as "C1" of the "H1" domain) typically extends from about amino acid 118 to about amino acid 215 (EU numbering system). The "hinge region" is generally defined as the region corresponding to Glu216 to Pro230 of human IgG1 in IgG (Burton, Molec. Immunol. 22: 161-206 (1985)). The hinge regions of other IgG isotypes can be aligned with the IgG1 sequence by placing the first and last cysteine ​​residues that form the inter-heavy chain SS bond in the same position. The "CH2 domain" (also referred to as the "C2" domain) of the human IgG Fc domain typically extends from about amino acid 231 to about amino acid 340. The uniqueness of the CH2 domain is that it is not closely paired with another domain. Instead, two N-connected branched carbohydrate chains are inserted between the two CH2 domains of the complete native IgG molecule. It is speculated that carbohydrates may provide an alternative to domain-domain pairing and help stabilize the CH2 domain. Burton, Molec Immunol. 22:161-206 (1985). A "CH3 domain" (also called a "C3" domain) comprises a stretch of residues C-terminal to the CH2 domain in the Fc domain (i.e., from about amino acid residue 341 of the antibody sequence to the C-terminus, typically at amino acid residue 446 or 447 in IgG).

[0080] The term "Fc domain" or "fragment crystallizable region" herein is used to define the C-terminal region of an immunoglobulin heavy chain, including a native sequence Fc domain and a variant Fc domain. Although the boundaries of the Fc domain of an immunoglobulin heavy chain may vary, the human IgG heavy chain Fc domain is generally defined as extending from an amino acid residue at position Cys226 or from Pro230 to its carboxyl terminus. The C-terminal lysine (residue 447 according to the EU numbering system) of the Fc domain can be removed, for example, during the production or purification of an Fc-containing protein, or can be removed by recombinant engineering encoding a nucleic acid containing an Fc protein. Therefore, a composition containing an Fc protein can include an Fc protein population containing all K447 residues removed, an Fc protein population containing an Fc protein population not removed from a K447 residue, and an Fc protein population containing an Fc protein containing a K447 residue and a mixture of Fc proteins containing Fc proteins not containing K447 residues. Suitable native sequence Fc domains for use in the Fc-containing proteins described herein may include human IgGl, IgG2 (IgG2A, IgG2B), IgG3, and IgG4.

[0081] "Percentage (%) of amino acid sequence identity" or "homology" associated with the polypeptide sequences identified herein is defined as the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in the compared polypeptides after sequence alignment with any conservative substitutions considered as part of the sequence identity. Alignment for determining percentage of amino acid sequence identity can be achieved by various means within the scope of those skilled in the art (e.g., 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 measuring alignment, including any algorithm required to achieve maximum alignment over the full length of the compared sequences. However, for purposes herein, % 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).

[0082] As used herein, the term "pharmaceutically effective amount" of a G-CSF dimer molecule formulation refers to an amount of the formulation that provides a therapeutic effect in an administration regimen.

[0083] As used herein, the terms "effective amount" and "pharmaceutically effective amount" refer to a sufficient amount of an agent to provide a desired biological outcome. The outcome can be a reduction (e.g., a reduction of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%) and / or alleviation of the signs, symptoms, or causes of a disease or condition, or any other desired change in a biological system. As will be appreciated by one of ordinary 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.

[0084] As used herein, the terms "pharmaceutical formulation" or "pharmaceutical composition" are used interchangeably herein and refer to a preparation which is in a form which permits the biological activity of the active ingredients contained therein to be effective, and which contains no other ingredients which are unacceptably toxic to a subject to which the formulation is administered.

[0085] A "sterile" preparation is sterile or substantially free of viable microorganisms and their spores.

[0086] As used herein, the term "absolute neutrophil count (ANC)" is a measure of the number of neutrophils present in the blood. Neutrophils are a type of white blood cell that plays a central role in the immune system's defense against infection, particularly bacterial infection. They are the most abundant type of white blood cell in most mammals and are an important part of the innate immune system.

[0087] As used herein, "bone marrow suppression" refers to the suppression of one or more components of hematopoiesis, which is manifested as abnormal levels of one or more cell types as products of the process. For a review of hematopoiesis and hematopoietic cell characteristics, see CLINICAL IMMUNOLOGY: PRINCIPLES AND Practice, Vol. 1, Ch. 2, pp. 15-24 (Lewis and Harriman, eds. Mosby-Year Book, Inc. 1996) (Clinical Immunology: Principles and Practice, Vol. 1, Chapter 2, pp. 15-24 (Lewis and Harriman, eds. Mosby-Year Book, Inc. 1996)), which are incorporated herein by reference. In general, bone marrow suppression refers to a reduction in white blood cell (WBC) and / or platelet counts. In a more specific sense, bone marrow suppression also refers to the suppression of one or more of the following hematopoietic cells: B cells, T cells, natural killer cells, dendritic cells, macrophages, neutrophils, eosinophils, basophils, mast cells and platelets. Thus, "bone marrow restoration," on the other hand, is the opposite of bone marrow suppression. A "bone marrow suppressive agent" or "bone marrow suppressive therapy" is a drug or therapy that is capable of inducing bone marrow suppression.

[0088] As used herein, the term "neutropenia" is defined as a condition characterized by a low number of neutrophils. Normal is defined as an absolute neutrophil count greater than or equal to 2.0 × 10 9 / L. Grade 1 is defined as an absolute neutrophil count of 1.5 × 10 9 / L and 1.999×10 9 Neutropenia between 1.0 × 10 / L. Grade 2 is defined as an absolute neutrophil count of 1.0 × 10 9 / L to 1.499×10 9 Neutropenia is defined as absolute neutrophil counts between 0.5 × 10 / L and grade 3 is defined as absolute neutrophil counts between 0.5 × 10 9 / L to 0.999×10 9 Grade 4 (severe neutropenia) is defined as an absolute neutrophil count of less than 0.5 × 10 9The terms “severe neutropenia” and “grade 4 neutropenia” are used interchangeably.

[0089] The term "febrile neutropenia" (FN) is defined as a single oral temperature ≥38.3°C (101°F) or a temperature ≥38.0°C (100.4°F) for 1 hour, accompanied by neutropenia, usually categorized as an ANC <500 cells / mm 3 Or ANC is expected to drop to < 500 cells / mm in the next 48 hours 3 This is a serious complication that often occurs in people undergoing cancer treatment, especially chemotherapy.

[0090] "Subjects" or "individuals" contemplated for administration include, but are not limited to, humans (e.g., males or females of any age group, such as pediatric subjects (e.g., infants, children, teenagers) or adult subjects (e.g., young, middle-aged, or elderly)) and / or non-human animals, such as primates (e.g., cynomolgus monkeys, rhesus monkeys), cows, pigs, horses, sheep, goats, rodents, cats, and / or dogs, among other mammals. 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 gender. In some embodiments, the individual suffers from cancer.

[0091] The terms "disease," "disorder," and "condition" are used interchangeably herein.

[0092] As used herein, "treatment" and "treating" are methods of obtaining beneficial or desired results (including clinical results). For the 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 caused by the disease (e.g., fever, infection, ANC reduction), reducing the scope of the disease (e.g., reducing the grade of neutropenia), shortening the duration of the disease, stabilizing the disease (e.g., preventing or delaying the deterioration of the disease), preventing or delaying the recurrence of the disease, delaying or slowing the progression of the disease (e.g., progressing to the next level of neutropenia or febrile neutropenia), improving the disease state, providing relief of the disease (partial or complete), reducing the dose of one or more other drugs required to treat the disease, improving or improving the quality of life, increasing weight gain and / or prolonging survival. "Treatment" also includes reducing pathological consequences (e.g., ANC reduction, infection, fever), including the severity and incidence of pathological consequences. The method of the present application contemplates any one or more of these aspects of treatment. When the methods are used to treat cancer, the methods, in some embodiments, can also prevent or slow the spread of cancer (eg, metastasis) and / or reduce pathological consequences (eg, tumor volume).

[0093] As used herein, "reference" refers to any sample, standard or level used for comparison purposes. The reference can be obtained from a healthy and / or non-diseased sample. In some instances, the reference can be obtained from an untreated sample. In some instances, the reference is obtained from an individual's non-diseased or untreated sample. In some instances, the reference is obtained from one or more healthy individuals who are not individuals or patients.

[0094] As used herein, "delaying the progression of a disease" means delaying, hindering, slowing, retarding, stabilizing, inhibiting and / or postponing the progression of a disease. The length of time for such delay may vary, depending on the history of the disease and / or the individual being treated. As is known to those skilled in the art, a substantial or significant delay may actually include prevention, in that the individual will not develop the disease.

[0095] As used herein, "prevention" includes preventing the occurrence or recurrence of a disease in an individual who may be susceptible to the disease but has not yet been diagnosed with the disease.

[0096] It should be understood that the embodiments of the present invention described herein include "consisting of" and / or "consisting essentially of" the embodiments.

[0097] As used herein, reference to a value or parameter that is "not" generally means and describes a value or parameter "other than that value or parameter." For example, the method is not used to treat type X means that the method is used to treat cancer types other than X.

[0098] As used herein and in the appended claims, the singular forms "a," "or," and "the" include plural referents unless the context clearly dictates otherwise.

[0099] II. Pharmaceutical Compositions

[0100] In some embodiments, a pharmaceutical composition comprising a G-CSF molecule is provided. Any of the G-CSF molecules described herein can be used in a pharmaceutical composition, including but not limited to a G-CSF dimer (eg, any G-CSF dimer described herein). 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, such as a G-CSF-Fc dimer); (b) about 1 mM to about 50 mM of a buffer (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 syringe. In some embodiments, the volume of the pharmaceutical composition in the syringe is about 1 mL. In some embodiments, the syringe is disposable. In some embodiments, the syringe is sterile. In some embodiments, the G-CSF dimer comprises two monomer subunits, each monomer subunit comprising (or consisting essentially of or consisting of): the amino acid sequence of any one of SEQ ID NO: 6-10 (e.g., SEQ ID NO: 6). In some embodiments, the G-CSF dimer is abegrastim alpha.

[0101] In some embodiments, disclosed herein are pharmaceutical compositions comprising a G-CSF dimer (e.g., any G-CSF dimer described herein). In some embodiments, the G-CSF dimer comprises 2 monomer subunits, wherein each monomer subunit comprises a G-CSF monomer and a dimerization domain. In some embodiments, the G-CSF dimer has an extended 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 abegrastim alpha.

[0102] In some embodiments, the pharmaceutical composition comprises a buffer (e.g., sodium acetate) of about 1 mM to about 50 mM. In some embodiments, the pharmaceutical composition comprises a stabilizer (e.g., EDTA) of about 0.1 mM to about 20 mM. In some embodiments, the pharmaceutical composition comprises a tonicity agent (e.g., sorbitol or sucrose) of about 1% (w / v) to about 15% (w / v), for example, about 1% (w / v) to about 10% (w / v). In some embodiments, the pharmaceutical composition comprises a surfactant (e.g., polysorbate 20) of about 0.001% (w / v) to about 0.1% (w / v). In some embodiments, 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.

[0103] Typically, the G-CSF dimer in the pharmaceutical composition remains soluble at a concentration of about 1 mg / mL to about 100 mg / mL, remains stable under isotonic storage conditions, and exhibits improved stability compared to currently available G-CSF formulations. In some embodiments, the pharmaceutical composition comprises at least 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 of any one of the G-CSF dimers. In some embodiments, the pharmaceutical composition comprises about 1 mg / mL to about 100 mg / mL of G-CSF dimer, for example, 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 comprises about 18 mg / mL to about 22 mg / mL of G-CSF dimer, for example, about 20 mg / mL of G-CSF-dimer.

[0104] Buffer can include but not limited to citrate, phosphate, acetate, succinate, tartrate, maleate, HEPES, Tris, N,N-bicine (Bicine), glycine, N-glycylglycine, carbonate, glycylglycine, lysine, arginine, histidine and / or its mixture.In some embodiments, buffer is sodium acetate.In some embodiments, pharmaceutical composition comprises about 1mM to about 50mM sodium acetate, for example, about 1mM to about 40mM, about 1mM to about 30mM, about 5mM to about 25mM, about 5mM to about 20mM or about 10mM of any one of sodium acetate.

[0105] In some embodiments, in the pharmaceutical compositions described herein, sodium acetate is a buffering agent that can be used to maintain the pH of the pharmaceutical composition at about pH 4.2 to about pH 6.2, such as 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), such as about pH 5.2.

[0106] Stabilizers can be added to improve the stability of pharmaceutical compositions described herein and extend their shelf life. 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), ethylene glycol bis-(2-aminoethyl)-N, N, N', N'-tetraacetic acid (EGTA), nitrilotriacetic acid (NTA), metal ion stabilizers and citrate. In some embodiments, stabilizer is EDTA. In some embodiments, the pharmaceutical composition comprises about 0.1 mM to about 20 mM EDTA, such as 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 can be used as a chelating agent, preservative, or stabilizer to prevent the pharmaceutical composition from catalyzing oxidation reactions during processing and storage.

[0107] In some embodiments, the pharmaceutical composition comprises one or more tonicity agents. Tonicity agents reduce (e.g., reduce at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% of any one) local irritation caused by the pharmaceutical preparation / composition by preventing osmotic shock at the site of administration. Examples of tonicity 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). In some embodiments, the tonicity agent is sorbitol. In some embodiments, the pharmaceutical composition comprises about 1% (w / v) to about 15% (w / v) sorbitol, such as 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) of any one of sorbitol. In some embodiments, the tonicity agent is sucrose. In some embodiments, the pharmaceutical composition comprises about 1% (w / v) to about 15% (w / v) sucrose, for example, about 5% (w / v) to about 15% (w / v), about 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), about 9% (w / v) to about 14% (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) sucrose.

[0108] Nonionic surfactants, such as polysorbates, including polysorbate 20 and polysorbate 80; Poloxamers, including Poloxamers 184 and 188; Polyols; and other ethylene / polypropylene block polymers stabilize the formulation and prevent protein adsorption by reducing interfacial interactions during processing and storage. In some embodiments, the surfactant is selected from 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), Polysorbate 20 at any of about 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).

[0109] Exemplified herein are G-CSF dimer pharmaceutical compositions wherein the G-CSF dimer is soluble at high protein concentrations and is stable during prolonged storage.

[0110] 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 (e.g., about 5 mg / mL to about 50 mg / mL, e.g., about 20 mg / mL) of a 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 buffer (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); (d) about 1% (w / v) to about 15% (w / v) (e.g., about 1% (w / v) to about 20% (w / v) of a stabilizer (e.g., EDTA); v) to about 10% (w / v), e.g., about 5% (w / v) to about 9% (w / v)) of a tonicity 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), e.g., about 0.01% (w / v)) of a surfactant (e.g., polysorbate 20 or polysorbate 80); wherein 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); wherein the G-CSF dimer comprises two monomer subunits, and wherein each monomer subunit comprises a G-CSF monomer and a dimerization domain. 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 (e.g., about 5 mg / mL to about 50 mg / mL, e.g., about 20 mg / mL) of a 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 buffer (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); (d) about 1% (w / v) to about 15% (w / v) (e.g., about 1% (w / v) to about 10% (w / v) of a dihydrotestosterone; (e.g., about 1% (w / v) to about 20% (w / v) of a dihydrotestosterone; (w / v), such as about 5% (w / v) to about 9% (w / v) of a tonicity 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), such as about 0.01% (w / v)) of a surfactant (e.g., polysorbate 20 or polysorbate 80); wherein 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); wherein the G-CSF dimer comprises two monomer subunits, and wherein each monomer subunit comprises 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 by an optional linker (e.g., 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 a fragment or variant thereof. In some embodiments, the Fc fragment comprises an amino acid sequence selected from SEQ ID NO: 2-5. In some embodiments, the G-CSF monomer is N-terminal relative to the dimerization domain within each monomer subunit. In some embodiments, each monomer subunit comprises (or consists essentially of, or consists of): an amino acid sequence of any one of SEQ ID NO: 6-10 or a variant thereof, wherein the variant has at least about 90% sequence identity with the amino acid sequence of any one of SEQ ID NO: 6-10. 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 (e.g., about 5 mg / mL to about 50 mg / mL, e.g., about 20 mg / mL) of a 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 buffer (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); (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) to about 9% (w / v) of a tonicity 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), e.g., about 0.01% (w / v)) of a surfactant (e.g., polysorbate 20 or polysorbate 80); wherein 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); wherein the G-CSF dimer comprises two monomer subunits, each monomer subunit comprising (or consisting essentially of, or consisting of): the amino acid sequence of any one 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) 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) (w / v) to about 10% (w / v), e.g., about 5% (w / v)) of sorbitol; 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); wherein 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); wherein the G-CSF dimer comprises two monomer subunits, each monomer subunit comprising (or consisting essentially of, or consisting of): the amino acid sequence of any one 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) 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) of (w / v) to about 15% (w / v), e.g., about 9% (w / v)) of 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)) of a surfactant (e.g., polysorbate 20 or polysorbate 80); wherein 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); wherein the G-CSF dimer comprises two monomer subunits, each monomer subunit comprising (or consisting essentially of, or consisting of): the amino acid sequence of any one of SEQ ID NOs: 6-10 (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 of a 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; wherein the pH of the pharmaceutical composition is about 5.0 to about 5.4 (e.g., about 5.2); wherein the G-CSF dimer comprises two monomer subunits, each monomer subunit comprising (or consisting essentially of, or consisting of): an amino acid sequence of any one of SEQ ID NOs: 6-10 (e.g., SEQ ID NO: 6). In some embodiments, the G-CSF dimer is abegrastim alpha. In some embodiments, 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 disposable. In some embodiments, the syringe is sterile.

[0111] 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, such as abegrastim α); (b) about 1 mM to about 50 mM (e.g., about 1 mM to about 30 mM, such as about 10 mM) of a buffer (e.g., sodium acetate); (c) about 0.1 mM to about 20 mM (e.g., about 0.1 mM to about 10 mM, such as about 1 mM) of a stabilizer (e.g., EDTA); (d) about 1% (w / v) to about 15% (w / v) of a succinate; v) (e.g., about 1% (w / v) to about 10% (w / v), e.g., about 5% (w / v) to about 9% (w / v)) of a tonicity 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), e.g., about 0.01% (w / v)) of a surfactant (e.g., polysorbate 20 or polysorbate 80); wherein 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, 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, such as abegrastim α); (b) about 1 mM to about 50 mM (e.g., about 1 mM to about 30 mM, such as 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, such as about 1 mM) of EDTA; (d) about 1% (w / v) to about 15% (w / v) of EDTA. / v) (e.g., about 1% (w / v) to about 10% (w / v), e.g., about 5% (w / v)) of sorbitol; 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); wherein 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, 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, such as abegrastim α); (b) about 1 mM to about 50 mM (e.g., about 1 mM to about 30 mM, such as 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, such as about 1 mM) of EDTA; (d) about 1% (w / v) to about 15% (w / v) of EDTA. (e.g., about 1% (w / v) to about 10% (w / v), such as about 9% (w / v) of 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), such as about 0.01% (w / v) of polysorbate 20 or polysorbate 80 (e.g., polysorbate 20); wherein 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 monomer subunits, each monomer subunit comprising (or consisting essentially of, or consisting of): the amino acid sequence of any one of SEQ ID NOs: 6-10 (e.g., SEQ ID NO: 6). In some embodiments, the G-CSF dimer is abegrastim alfa. In some embodiments, 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 disposable. In some embodiments, the syringe is sterile.

[0112] 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 such as abegrastim alpha); (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 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 a G-CSF dimer (e.g., a G-CSF-Fc dimer, such as abegrastim α); (b) about 1 mM to about 50 mM of sodium acetate; (c) about 0.1 mM to about 20 mM of EDTA; (d) about 1% (w / v) to about 10% (w / v) of sorbitol; and (e) about 0.001% (w / v) to about 0.1% (w / v) of polysorbate 20. 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, such as abegrastim alpha); (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, a pharmaceutical composition is provided, comprising (or consisting essentially of, or consisting of): (a) about 1 mg / mL to about 100 mg / mL (e.g., about 20 mg / mL) of a G-CSF dimer (e.g., a G-CSF-Fc dimer, such as abegrastim α); (b) about 1 mM to about 50 mM of sodium acetate; (c) about 0.1 mM to about 20 mM of EDTA; (d) about 1% (w / v) to about 15% (w / v) of sucrose; and (e) about 0.001% (w / v) to about 0.1% (w / v) of 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 monomer subunits, each monomer subunit comprising (or consisting essentially of, or consisting of): the amino acid sequence of any one of SEQ ID NOs: 6-10 (e.g., SEQ ID NO: 6). In some embodiments, the G-CSF dimer is abegrastim alpha. In some embodiments, the pharmaceutical composition is contained in a syringe. In some embodiments, the volume of the pharmaceutical composition within the syringe is about 1 mL.In some embodiments, the syringe is single-use. In some embodiments, the syringe is sterile.

[0113] 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, such as abegrastim alpha); (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, wherein 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, a pharmaceutical composition is provided comprising (or consisting essentially of, or consisting 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 such as abegrastim alpha); (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, wherein 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, 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, such as abegrastim alpha); (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, wherein 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, a pharmaceutical composition is provided comprising (or consisting essentially of, or consisting 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 such as abegrastim alpha); (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, wherein 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 monomer subunits, each subunit comprising (or consisting essentially of, or consisting of): the amino acid sequence of any one of SEQ ID NOs: 6-10 (e.g., SEQ ID NO: 6). In some embodiments, the G-CSF dimer is abegrastim alpha. In some embodiments, the pharmaceutical composition is contained in a syringe. In some embodiments, the volume of the pharmaceutical composition within the syringe is about 1 mL. In some embodiments, the syringe is disposable. In some embodiments, the syringe is sterile.

[0114] 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, such as abegrastim alpha); (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, wherein the pH of the pharmaceutical composition is 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 (or consisting essentially of, or consisting of): (a) about 5 mg / mL to about 50 mg / mL (e.g., about 20 mg / mL) of a G-CSF dimer (e.g., a G-CSF-Fc dimer, such as abegrastim alpha); (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, wherein the pH of the pharmaceutical composition is 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 (or consisting essentially of, or consisting of): (a) a G-CSF dimer (e.g., a G-CSF-Fc dimer, such as abegrastim alpha); (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, wherein the pH of the pharmaceutical composition is 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 (or consisting essentially of, or consisting of): (a) about 5 mg / mL to about 50 mg / mL (e.g., about 20 mg / mL) of a G-CSF dimer (e.g., a G-CSF-Fc dimer, such as abegrastim alpha); (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, wherein the pH of the pharmaceutical composition is about 4.8 to about 5.8 (e.g., about 5.0 to about 5.4, or about 5.2).In some embodiments, the G-CSF dimer comprises two monomer subunits, each subunit comprising (or consisting essentially of, or consisting of) the amino acid sequence of any one of SEQ ID NOs: 6-10 (e.g., SEQ ID NO: 6). In some embodiments, the G-CSF dimer is abegrastim alpha. In some embodiments, the pharmaceutical composition is contained in a syringe. In some embodiments, the volume of the pharmaceutical composition within the syringe is about 1 mL. In some embodiments, the syringe is disposable. In some embodiments, the syringe is sterile.

[0115] 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, such as abegrastim alpha); (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, wherein 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, a pharmaceutical composition is provided comprising (or consisting essentially of, or consisting of): (a) about 20 mg / mL of a G-CSF dimer (e.g., a G-CSF-Fc dimer, such as abegrastim alpha); (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, wherein 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, 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, such as abegrastim alpha); (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, wherein 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, a pharmaceutical composition is provided, comprising (or consisting essentially of, or consisting of): (a) about 20 mg / mL of a G-CSF dimer (e.g., a G-CSF-Fc dimer, such as abegrastim alpha); (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, wherein 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 monomer subunits, each of which comprises (or consists essentially of, or consists of): an amino acid sequence of any one of SEQ ID NOs: 6-10 (e.g., SEQ ID NO: 6). In some embodiments, the G-CSF dimer is abegrastim alpha. In some embodiments, 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 disposable. In some embodiments, the syringe is sterile.

[0116] 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, such as abegrastim alpha); (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, wherein the pH of the pharmaceutical composition is about 5.0 to about 5.4 (e.g., about 5.2). In some embodiments, a pharmaceutical composition is provided, comprising (or consisting essentially of, or consisting of): (a) about 20 mg / mL of a G-CSF dimer (e.g., a G-CSF-Fc dimer, such as abegrastim α); (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, wherein the pH of the pharmaceutical composition is about 5.0 to about 5.4 (e.g., about 5.2). In some embodiments, the G-CSF dimer comprises two monomer subunits, each of which comprises (or consists essentially of, or consists of): the amino acid sequence of any one of SEQ ID NOs: 6-10 (e.g., SEQ ID NO: 6). In some embodiments, a pharmaceutical composition is provided, comprising (or consisting essentially of, or consisting of): (a) abegrastim alpha; (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, wherein the pH of the pharmaceutical composition is about 5.0 to about 5.4 (e.g., about 5.2). In some embodiments, a pharmaceutical composition is provided, comprising (or consisting essentially of, or consisting of): (a) about 20 mg / mL abegrastim alpha; (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, wherein the pH of the pharmaceutical composition is about 5.0 to about 5.4 (e.g., about 5.2). In some embodiments, 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 disposable. In some embodiments, the syringe is sterile.

[0117] 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, such as abegrastim alpha); (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, wherein the pH of the pharmaceutical composition is about 5.0 to about 5.4 (e.g., about 5.2). In some embodiments, a pharmaceutical composition is provided, comprising (or consisting essentially of, or consisting of): (a) about 20 mg / mL of a G-CSF dimer (e.g., a G-CSF-Fc dimer, such as abegrastim α); (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, wherein the pH of the pharmaceutical composition is about 5.0 to about 5.4 (e.g., about 5.2). In some embodiments, the G-CSF dimer comprises two monomer subunits, each of which comprises (or consists essentially of, or consists of): the amino acid sequence of any one of SEQ ID NOs: 6-10 (e.g., SEQ ID NO: 6). In some embodiments, a pharmaceutical composition is provided, comprising (or consisting essentially of, or consisting of): (a) abegrastim alpha; (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, wherein the pH of the pharmaceutical composition is about 5.0 to about 5.4 (e.g., about 5.2). In some embodiments, a pharmaceutical composition is provided, comprising (or consisting essentially of, or consisting of): (a) about 20 mg / mL abegrastim alpha; (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, wherein the pH of the pharmaceutical composition is about 5.0 to about 5.4 (e.g., about 5.2). In some embodiments, 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 disposable. In some embodiments, the syringe is sterile.

[0118] In some embodiments, a pharmaceutical composition is provided, comprising (or consisting essentially of, or consisting of): (a) about 20 mg / mL of abegrastim alpha; (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, wherein the pH of the pharmaceutical composition is about 5.2. Such a pharmaceutical composition is also referred to herein as an "abegrastim alpha pharmaceutical composition". In some embodiments, a pharmaceutical composition is provided, comprising (or consisting essentially of, or consisting of): (a) 20 mg / mL of abegrastim alpha; (b) 10 mM sodium acetate; (c) 1 mM EDTA; (d) 5% (w / v) sorbitol; and (e) 0.01% (w / v) polysorbate 20, wherein the pH of the pharmaceutical composition is 5.2. In some embodiments, 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 disposable. In some embodiments, the syringe is sterile. In some embodiments, the pharmaceutical composition is a clear, colorless, preservative-free solution supplied in a prefilled single-dose syringe with a 27-gauge, 1 / 2-inch needle and an UltraSafe Passive TM Needle shield, containing 20 mg of abegrastim alfa. In some embodiments, each syringe contains about 20 mg of abegrastim alfa in a sterile, clear, colorless, preservative-free solution (about pH 5.2) containing acetate (about 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 abegrastim alfa in a sterile, clear, colorless, preservative-free solution (pH 5.2) containing 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.

[0119] 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, such as abegrastim alpha); (b) sodium; (c) acetate; (d) EDTA; (e) sorbitol; and (f) polysorbate 20 or polysorbate 80 (e.g., polysorbate 20). 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, such as abegrastim alpha); (b) sodium; (c) acetate; (d) EDTA; (e) sucrose; and (f) polysorbate 20 or polysorbate 80 (e.g., polysorbate 20). 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, such as abegrastim alpha); (b) sodium acetate; (c) EDTA; (d) sorbitol; and (e) polysorbate 20 or polysorbate 80 (e.g., polysorbate 20). 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, such as abegrastim alpha); (b) sodium acetate; (c) EDTA; (d) sucrose; and (e) polysorbate 20 or polysorbate 80 (e.g., 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 pharmaceutical composition further comprises water. In some embodiments, the G-CSF dimer comprises two monomer subunits, each monomer subunit comprising (or consisting essentially of, or consisting of): the amino acid sequence of any one of SEQ ID NOs: 6-10 (e.g., SEQ ID NO: 6). In some embodiments, the G-CSF dimer is abegrastim alpha. In some embodiments, the pharmaceutical composition comprises (or consists essentially of, or consists of): about 20 mg of abegrastim alpha, about 0.6 mg of acetate, about 0.29 mg of EDTA, about 0.1 mg of polysorbate 20, about 0.23 mg of sodium, and about 50 mg of sorbitol. In some embodiments, the pharmaceutical composition is lyophilized. In some embodiments, the pharmaceutical composition is in about 1 mL of solution (e.g., in water). In some embodiments, the pH of the pharmaceutical composition is about 5.2.

[0120] In some embodiments, a syringe is also provided, comprising any pharmaceutical composition described herein (e.g., a G-CSF-Fc dimer pharmaceutical composition, such as an abegrastim α pharmaceutical composition). In some embodiments, the volume of the pharmaceutical composition in the syringe is about 1 mL. In some embodiments, the syringe is disposable. In some embodiments, the syringe is sterile. In some embodiments, the prefilled syringe does not carry a scale mark and is only used to deliver the entire contents of the syringe (20 mg / 1 mL) for direct administration to an individual (e.g., an adult). In some embodiments, the needle cap of the prefilled syringe comprises natural rubber latex.

[0121] Characteristics of the pharmaceutical composition

[0122] 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%, 96%, 97%, 98%, 99% or more) of G-CSF dimer (e.g., G-CSF-Fc dimer, such as abegrastim alpha) as assessed by SE-HPLC. In some embodiments, the purity is measured before storage. In some embodiments, the 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.

[0123] In some embodiments, the pharmaceutical compositions described herein are characterized by extended stability. In some embodiments, the pharmaceutical compositions comprising a G-CSF dimer (e.g., a G-CSF-Fc dimer, such as abegrastim alpha) 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 any of longer periods. In some embodiments, the pharmaceutical compositions comprising a G-CSF dimer (e.g., a G-CSF-Fc dimer, such as abegrastim alpha) 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 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 any one of longer periods. In some embodiments, the pharmaceutical composition is stable for up to about 6 months at 25 ± 2 ° C, for example, up to about 3 months (for example, stable after storage for 3, 2 or 1 months). In some embodiments, stability is assessed after storing the liquid pharmaceutical composition under suitable storage conditions. In some embodiments, after storing the lyophilized pharmaceutical composition under suitable storage conditions, the liquid pharmaceutical composition reconstructed by the lyophilized pharmaceutical composition is subjected to stability assessment. In some embodiments, after storing the frozen pharmaceutical composition under suitable storage conditions, the liquid pharmaceutical composition reconstructed by the frozen pharmaceutical composition is subjected to stability assessment.

[0124] In some embodiments, storage conditions include storage at about 2°C to about 8°C. In some embodiments, storage conditions include storage at 25±2°C. In some embodiments, 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 any one of the longer periods. In some embodiments, storage conditions include storage for up to about 36 months, for example, up to about 6 months, or up to about 3 months. In some embodiments, storage conditions include storage for up to about 36 months at about 2°C to about 8°C. In some embodiments, storage conditions include storage for up to about 3 months at 25±2°C. In some embodiments, storage conditions include protection from light. In some embodiments, storage conditions include avoiding shaking. In some embodiments, storage conditions include avoiding freezing. In some embodiments, storage conditions include freezing and then thawing at about 2°C to about 8°C before use. In some embodiments, storage conditions include avoiding freezing more than once. In some embodiments, storage conditions include storage at 25±2°C for no more than about 48 hours.

[0125] In some embodiments, the pharmaceutical composition (e.g., a G-CSF-Fc dimer pharmaceutical composition, such as an abegrastim alfa pharmaceutical composition) has excellent freeze-thaw stability, for example, is stable after being frozen at about -20°C and thawed at room temperature for at least 6 (e.g., 6, 7, 8, or more) cycles.

[0126] In some embodiments, the pharmaceutical composition (e.g., a G-CSF-Fc dimer pharmaceutical composition, such as an abegrastim alpha pharmaceutical composition) is within a clinically acceptable range after storage. In some embodiments, the pharmaceutical composition comprises at least about 96% (e.g., at least about any one of 97%, 98%, 99%, or 100%) of G-CSF dimer as measured by size exclusion chromatography (SEC) after storage. In some embodiments, the pharmaceutical composition comprises at least about 95.5% (e.g., at least about any one of 96%, 97%, 98%, 99%, or 100%) of G-CSF dimer as measured by rCE-SDS after storage. In some embodiments, the pharmaceutical composition comprises at least about 94% (e.g., at least about any one of 95%, 96%, 97%, 98%, 99%, or 100%) of G-CSF dimer as measured by nrCE-SDS after storage. In some embodiments, the pharmaceutical composition comprises no more than about 1.5% (e.g., no more than about 1.4%, 1.3%, 1.2%, 1.1%, 1.0%, 0.8%, 0.6%, 0.4%, 0.1%, or less) of HMWS as measured by rCE-SDS after storage. In some embodiments, the pharmaceutical composition comprises no more than about 1.5% (e.g., no more than about 1.4%, 1.3%, 1.2%, 1.1%, 1.0%, 0.8%, 0.6%, 0.4%, 0.1%, or less) of HMWS as measured by nrCE-SDS after storage. In some embodiments, the pharmaceutical composition comprises an acidic region of about 27.2% to about 39.0% (e.g., about 30% to about 40%, such as about 32% to about 37%), a main peak of about 36.5% to about 60.1% (e.g., about 45% to about 55%, such as about 47% to about 55%), and a basic region of about 7.2% to about 30% (e.g., about 10% to about 20%, such as about 11% to 18%) as measured by icIEF after storage. 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%) of potency (e.g., potency of G-CSF) after storage. In some embodiments, the pharmaceutical composition retains about 70% to about 130% (e.g., any of about 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, or 130%) of potency (e.g., potency of G-CSF) after storage, for example, as determined by a cell proliferation assay. In some embodiments, the concentration of the pharmaceutical composition (e.g., G-CSF dimer concentration) changes 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) after storage.In some embodiments, storage conditions include storage at about 2°C to about 8°C for at least about 36 months. In some embodiments, storage conditions include storage at 25±2°C for at least about 1 month (e.g., at least about 2 months). 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.

[0127] 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 includes one or more of the following: i) promoting (e.g., at least about any of 10%, 20%, 50%, 1-fold, 2-fold, 10-fold, 20-fold or greater) the growth and / or differentiation of blood cells (e.g., neutrophils); ii) enhancing (e.g., at least about any of 10%, 20%, 50%, 1-fold, 2-fold, 10-fold, 20-fold or greater) an immune response (e.g., defense against bacterial infection); iii) activating mature neutrophils (e.g., participating in an immune response, or combining with other hematopoietic growth factors such as stem cell factor, Flt-3 ligand and GM-CSF); synergistically to perform hematopoietic function); iv) improve (e.g., at least about any of 10%, 20%, 50%, 1-fold, 2-fold, 10-fold, 20-fold or more) hematopoietic function; v) increase (e.g., at least about any of 10%, 20%, 50%, 1-fold, 2-fold, 10-fold, 20-fold or more) neutrophil count; vi) promote (e.g., at least about any of 10%, 20%, 50%, 1-fold, 2-fold, 10-fold, 20-fold or more) neutrophil mobilization; and vii) reduce (e.g., at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%) the mobilization of peripheral blood progenitor cells.

[0128] In some embodiments, the pharmaceutical composition (e.g., liquid pharmaceutical composition) has an appearance of one or more of the following characteristics: i) transparent, ii) colorless to slightly yellow, iii) substantially free of (e.g., no more than about 10%, e.g., no more than 8%, 5%, 2%, 1% or any one of less) visible particles. The appearance can 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. In some embodiments, the pharmaceutical composition is placed in a colorimetric test tube, and visible particles are checked with the naked eye under black and white backgrounds, respectively. In some embodiments, the pharmaceutical composition is compared to a colorimetric standard of a white background to assess color.

[0129] In some embodiments, the pharmaceutical composition is characterized by fewer and / or smaller particles. In some embodiments, the pharmaceutical composition comprises ≤6000 particles with a diameter ≥10 μm per vial and ≤600 particles with a diameter ≥25 μm per vial. In some embodiments, the pharmaceutical composition is transferred to a vial and a particle size measurement system (e.g., ACCUSIZER 780SIS) is used to check the particles in the vial. The particle properties of the pharmaceutical composition can 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.

[0130] In some embodiments, the pharmaceutical compositions described herein (e.g., G-CSF-Fc dimer pharmaceutical compositions, such as abegrastim alpha 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, such as about 1 to about 45 mPa-S, about 1 to 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 2 mPa-S, between about 5 ° C and about 25 ° C. For example, in some embodiments, the viscosity of the pharmaceutical composition at about 25 ° C is about 1-2 mPa-S. In some embodiments, the pharmaceutical composition is isotonic. The viscosity characteristics of the pharmaceutical composition can 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.

[0131] In some embodiments, the osmolality of a pharmaceutical composition described herein (e.g., a G-CSF-Fc dimer pharmaceutical composition, such as an abegrastim alpha pharmaceutical composition) ranges from about 100 to about 500 mOsm / kg, such as 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 profile of the pharmaceutical composition can 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, storage conditions include storage at 25±2°C for up to about 3 months.

[0132] In some embodiments, the pharmaceutical compositions described herein (e.g., G-CSF-Fc dimer pharmaceutical compositions, such as abegrastim alpha pharmaceutical compositions) are characterized by a low percentage of protein aggregation (e.g., before and / or after storage). In some embodiments, the percentage of total protein aggregation is less than about 5%, for example, less than any of 4%, 3%, 2%, 1%, 0.5%, 0.2%, 0.1%, or less. In some embodiments, the percentage of total protein aggregation measured by SEC is less than about 2%. In some embodiments, storage conditions include storage at about 2°C to about 8°C for at least about 36 months. In some embodiments, storage conditions include storage at 25±2°C for at least about 1 month (e.g., at least about 2 months). 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.

[0133] In some embodiments, the pharmaceutical composition (e.g., a G-CSF-Fc dimer pharmaceutical composition, such as an abegrastim alpha pharmaceutical composition) comprises no more than about 1.5% (e.g., no more than 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.8%, 0.5%, 0.1% or any less, such as no more than about 1%) of 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 no more than about 1.5% (e.g., no more than about 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.8%, 0.5%, 0.1% or any less, such as no more than about 1%) of 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 no more than about 1.5% HMWS as measured by rCE-SDS after storage for up to about 3 months at 25±2°C. In some embodiments, the pharmaceutical composition comprises no more than about 1.5% (e.g., no more than about 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.8%, 0.5%, 0.1% or any one of less) HMWS as measured by rCE-SDS after storage for at least about 36 months at about 2°C to about 8°C. In some embodiments, the pharmaceutical composition comprises no more than about 1.5% HMWS as measured by rCE-SDS after storage for up to about 36 months at about 2°C to about 8°C. In some embodiments, after storage at 25±2°C for at least about 1 month, the pharmaceutical composition comprises no more than about 1.5% (e.g., no more than about 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.8%, 0.5%, 0.1% or less) of HMWS as measured by nrCE-SDS. In some embodiments, after storage at 25±2°C for at least about 2 months, the pharmaceutical formulation comprises no more than about 1.5% (e.g., no more than about 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.8%, 0.5%, 0.1% or less) of HMWS as measured by nrCE-SDS. In some embodiments, after storage at 25±2°C for up to about 3 months, the pharmaceutical formulation comprises no more than about 1.5% of HMWS as measured by nrCE-SDS. In some embodiments, the pharmaceutical composition comprises no more than about 1.5% (e.g., no more than about any of 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.8%, 0.5%, 0.1%, or less) HMWS 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 no more than about 1.5% HMWS as measured by nrCE-SDS after storage for up to about 36 months at about 2° C. to about 8° C.

[0134] In some embodiments, the pharmaceutical composition (e.g., a G-CSF-Fc dimer pharmaceutical composition, such as an abegrastim alpha 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) of G-CSF dimer 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%) of G-CSF dimer, e.g., at least about 95.5% or at least about 96% of G-CSF dimer, after storage at 25±2°C for at least about 1 month as measured by rCE-SDS. 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%) of G-CSF dimer as measured by rCE-SDS after storage at 25±2°C for at least about 2 months, such as at least about 95.5% or at least about 96% of G-CSF dimer. In some embodiments, the pharmaceutical composition comprises at least about 90% of G-CSF dimer as measured by rCE-SDS after storage at 25±2°C for up to about 3 months, such as at least about 95.5% or at least about 96% of G-CSF dimer. 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%) of G-CSF dimer, e.g., at least about 95.5% of G-CSF dimer, as measured by rCE-SDS after storage at about 2°C to about 8°C for at least about 36 months. In some embodiments, the pharmaceutical composition comprises at least about 90% of G-CSF dimer, e.g., at least about 95.5% of G-CSF dimer, as measured by rCE-SDS after storage at about 2°C to about 8°C for up to about 36 months. In some embodiments, the pharmaceutical composition comprises at least about 90% (e.g., at least about any one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of G-CSF dimer, such as at least about 94% of G-CSF dimer, as measured by nrCE-SDS after storage at 25±2°C for at least about 1 month. In some embodiments, the pharmaceutical composition comprises at least about 90% (e.g., at least about any one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of G-CSF dimer, such as at least about 94% of G-CSF dimer, as measured by nrCE-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, such as at least about 94% G-CSF dimer, as measured by nrCE-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 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) G-CSF dimer, such as at least about 94% G-CSF dimer, as measured by nrCE-SDS after storage at about 2°C to about 8°C for at least about 36 months. In some embodiments, the pharmaceutical composition comprises at least about 90% G-CSF dimer, such as at least about 94% G-CSF dimer, as measured by nrCE-SDS after storage at about 2°C to about 8°C for up to about 36 months.

[0135] In some embodiments, the pharmaceutical composition (e.g., a G-CSF-Fc dimer pharmaceutical composition, such as an abegrastim alfa 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%) of G-CSF dimer, such as at least about 97% of G-CSF dimer, as measured by SEC. In some embodiments, the pharmaceutical composition comprises a major peak of 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) 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 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 relative to the total area of ​​all peaks when measured by SE-HPLC after storage for 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 up to about 3 months 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 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.

[0136] In some embodiments, the pharmaceutical composition (e.g., a G-CSF-Fc dimer pharmaceutical composition, such as an abegrastim alfa pharmaceutical composition) comprises less than about 5% (e.g., less than 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.5%, 1%, 0.5%, 0.1% or any less, such as 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 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 any less, such as 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. In some embodiments, when measured by SE-HPLC after storage for up to about 3 months at 25±2°C, the pharmaceutical composition comprises less than about 5% (e.g., less than about 2.5%) dimer relative to the total area of ​​all peaks. In some embodiments, when measured by SE-HPLC after storage for at least about 36 months at about 2°C to about 8°C, 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 any one of less, for example, less than about 2.5%) dimer relative to the total area of ​​all peaks. In some embodiments, when measured by SE-HPLC after storage for up to about 36 months at about 2°C to about 8°C, the pharmaceutical composition comprises less than about 5% (e.g., less than about 2.5%) dimer relative to the total area of ​​all peaks.

[0137] In some embodiments, the pharmaceutical composition (e.g., a G-CSF-Fc dimer pharmaceutical composition, such as an abegrastim alpha pharmaceutical composition) comprises no more than about 2% (e.g., no more than any one of 1.8%, 1.5%, 1%, 0.5%, 0.1% or less) of 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 comprises no more than about 2% (e.g., no more than any one of about 1.8%, 1.5%, 1%, 0.5%, 0.1% or less) of 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 comprises no more than about 2% of aggregates relative to the total area of ​​all peaks when measured by SE-HPLC after storage at 25±2°C for up to about 3 months. In some embodiments, the pharmaceutical composition comprises no more than about 2% (e.g., no more than about 1.8%, 1.5%, 1%, 0.5%, 0.1%, or less) of aggregates 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 no more than about 2% of aggregates 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.

[0138] In some embodiments, the pharmaceutical composition (e.g., liquid pharmaceutical composition) (e.g., G-CSF-Fc dimer pharmaceutical composition, e.g., abegrastim 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 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 after storage at 25±2°C for at least about 1 month. In some embodiments, the pharmaceutical composition (e.g., a liquid pharmaceutical composition) comprises about 27.2% to about 39.0% (e.g., about 30% to about 40%, such as about 32% to about 37%) of an acidic region, about 36.5% to about 60.1% (e.g., about 45% to about 55%, such as about 47% to about 55%) of a main peak, and about 7.2% to about 30% (e.g., about 10% to about 20%, such as about 11% to 18%) of a basic region as measured by icIEF after storage at 25±2°C for at least about 2 months. In some embodiments, the pharmaceutical composition (e.g., a liquid pharmaceutical composition) comprises about 27.2% to about 39.0% of an acidic region, about 36.5% to about 60.1% of a main peak, and about 7.2% to about 30% of a basic region as measured by icIEF after storage at 25±2°C for up to about 3 months. In some embodiments, the pharmaceutical composition (e.g., a liquid pharmaceutical composition) comprises about 27.2% to about 39.0% (e.g., about 30% to about 40%, such as about 32% to about 37%) of the acidic region, about 36.5% to about 60.1% (e.g., about 45% to about 55%, such as about 47% to about 55%) of the main peak, and about 7.2% to about 30% (e.g., about 10% to about 20%, such as about 11% to 18%) of the basic region as 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., a liquid pharmaceutical composition) comprises about 27.2% to about 39.0% of the acidic region, about 36.5% to about 60.1% of the main peak, and about 7.2% to about 30% of the basic region as measured by icIEF after storage at about 2°C to about 8°C for up to about 36 months.

[0139] In some embodiments, after storage at 25±2°C for at least about 1 month, the pharmaceutical composition (e.g., a G-CSF-Fc dimer pharmaceutical composition, such as an abegrastim alpha pharmaceutical composition) retains at least about 70% (e.g., at least about any one of 75%, 80%, 85%, 90%, 95%, 99%, or 100%) of potency (e.g., potency of G-CSF). In some embodiments, after storage at 25±2°C for at least about 2 months, the pharmaceutical composition retains at least about 70% (e.g., at least about any one of 75%, 80%, 85%, 90%, 95%, 99%, or 100%) of potency (e.g., potency of G-CSF). In some embodiments, after storage at 25±2°C for up to about 3 months, the pharmaceutical composition retains at least about 70% of potency (e.g., potency of G-CSF). 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%) of potency (e.g., potency of G-CSF) after storage for at least about 36 months at about 2°C to about 8°C. In some embodiments, the pharmaceutical composition retains at least about 70% of potency (e.g., potency of G-CSF) after storage for up to about 36 months at about 2°C to about 8°C.

[0140] In some embodiments, the concentration of a pharmaceutical composition (e.g., a G-CSF-Fc dimer pharmaceutical composition, such as an abegrastim alpha pharmaceutical composition) (e.g., G-CSF dimer concentration) changes 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, or less) after storage at 25±2°C for at least about 1 month. In some embodiments, the concentration of a pharmaceutical composition (e.g., G-CSF dimer concentration) changes 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, or less) after storage at 25±2°C for at least about 2 months. In some embodiments, the concentration of a pharmaceutical composition (e.g., G-CSF dimer concentration) changes by less than about 0.5 mg / mL after storage at 25±2°C for up to about 3 months. In some embodiments, the concentration of the pharmaceutical composition (e.g., G-CSF dimer concentration) changes 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, or less) after storage for at least about 36 months at about 2°C to about 8°C. In some embodiments, the concentration of the pharmaceutical composition (e.g., G-CSF dimer concentration) changes by less than about 0.5 mg / mL after storage for up to about 36 months at about 2°C to about 8°C.

[0141] In some embodiments, commercial batches of any pharmaceutical composition described herein (e.g., GCSF-Fc dimer pharmaceutical composition, such as abegrastim α pharmaceutical composition) are provided. "Commercial batches" as used herein refer to batch sizes of at least about 20 grams. Commercial batches are produced on a scale greater than experimental or laboratory scale. The increase in scale is associated with longer production times (including longer steps (such as evaporation steps) or longer holding times between steps). In some embodiments, the change in the pharmaceutical composition between batches (e.g., the amount of the pharmaceutical composition component, the pH value of the pharmaceutical composition, the purity of the pharmaceutical composition, and any one of the characteristics of the pharmaceutical composition (such as the composition before and after storage, stability, viscosity, osmotic pressure molar concentration, purity and / or potency)) does not exceed about 5% (e.g., no more than about 4%, 3%, 2%, 1%, 0.5%, 0.1% or less).

[0142] Also provided is a method of preparing any pharmaceutical composition described herein (e.g., a G-CSF-Fc dimer pharmaceutical composition, such as an abegrastim alpha pharmaceutical composition), comprising mixing the components of the pharmaceutical composition in the amounts described herein to prepare the pharmaceutical 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., G-CSF dimer) before mixing with the other components of the pharmaceutical composition. In some embodiments, the method further comprises testing (or verifying) one or more properties of the pharmaceutical composition before and / or after storage, such as composition, stability, viscosity, osmolarity, purity, and / or potency.

[0143] III. Methods for Validating Pharmaceutical Compositions

[0144] In another aspect, the present application provides a method for evaluating the suitability of any pharmaceutical composition described herein (e.g., a G-CSF-Fc dimer pharmaceutical composition, such as an abegrastim alpha pharmaceutical composition) for medical use in an individual (e.g., a human individual, such as a human suffering from cancer and / or neutropenia). Such a validated pharmaceutical composition is 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 (e.g., a human, such as a human suffering from cancer and / or neutropenia) is provided, wherein 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 dimer (e.g., a G-CSF-Fc dimer, such as an abegrastim alpha); (b) about 1 mM to about 50 mM of a buffer (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), 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), the method comprising measuring a quality control parameter of the pharmaceutical composition; and assessing the suitability of the pharmaceutical composition for medical use in an individual, wherein the measured quality control parameter within a quality control parameter threshold indicates the suitability of the pharmaceutical composition for medical use. In some embodiments, a method of assessing the suitability of a pharmaceutical composition for medical use in an individual (e.g., a human, e.g., a human with cancer and / or neutropenia) is provided, wherein the pharmaceutical composition comprises (or consists essentially of, or consists of): (a) about 20 mg / mL of a G-CSF dimer (e.g., a G-CSF-Fc dimer, e.g., abegrastim alpha); (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, wherein the pH of the pharmaceutical composition is about 5.2, the method comprising measuring a quality control parameter of the pharmaceutical composition; and assessing the suitability of the pharmaceutical composition for medical use in an individual, wherein the measured quality control parameter within a quality control parameter threshold indicates suitability of the pharmaceutical composition for medical use. In some embodiments, the G-CSF dimer comprises two monomer subunits, each monomer subunit comprising (or consisting essentially of, or consisting of): the amino acid sequence of any one of SEQ ID NOs: 6-10 (eg, SEQ ID NO: 6).In some embodiments, the G-CSF dimer is abegrastim alpha. The quality control parameter can be any one or a combination of a plurality of parameters (e.g., composition, viscosity, stability, osmolality, purity, or potency) of the pharmaceutical composition described herein, which are evaluated, 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 in "II. Pharmaceutical Compositions" above can be used for validation purposes herein. For one or more quality control parameters, acceptance criteria (e.g., quality control parameter thresholds), and related methods, see Example 8 and Table 14.

[0145] 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 is a transparent, colorless to slightly yellow solution, and substantially free of (e.g., no more than about 10%, for example no more than about 8%, 5%, 2%, 1% or any one of less) visible particles indicating that the pharmaceutical composition is suitable for medical use. In some embodiments, the pharmaceutical composition is placed in a colorimetric test tube and the visible particles are checked with the naked eye under black and white backgrounds, respectively. In some embodiments, the pharmaceutical composition is compared with a colorimetric standard of a white background to assess the 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, 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.

[0146] In some embodiments, the quality control parameters include the pH value of the pharmaceutical composition (e.g., before and / or after storage). In some embodiments, the pH of the pharmaceutical composition is about 5.0 to about 5.4 (or 5.2 ± 0.2, e.g., about 5.2), indicating that the pharmaceutical composition is suitable for medical use. In some embodiments, the pH is measured using a pH meter. 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, 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.

[0147] In some embodiments, the quality control parameters include the particle size and / or amount (e.g., particles per container) of 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 checked using a particle size measurement system (e.g., ACCUSIZER 780SIS). In some embodiments, ≤6000 particles with a diameter ≥10 μm per bottle and ≤600 particles with a diameter ≥25 μm per bottle indicate that the pharmaceutical composition is suitable for medical use. 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, 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.

[0148] In some embodiments, the quality control parameters include the composition of the pharmaceutical composition (for example, 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 components. In some embodiments, the method includes, for example, measuring the pH value of the pharmaceutical composition using a pH meter. Any suitable method for measuring the composition components can be used herein, including but not limited to spectrophotometer, mass spectrometry (MS), rCE-SDS, nrCE-SDS, r-SDS-PAGE, nr-SDS-PAGE, SEC, HPLC (for example, SE-HPLC, RP-HPLC), icIEF or light scattering detection (LSD). In some embodiments, if the concentration and / or pH of each component of the pharmaceutical composition is within about 5% (for example, about 4%, 3%, 2%, 1%, 0.5%, 0.1% or any one of lower) of the concentration and / or pH of each corresponding component described herein, it is indicated that the pharmaceutical composition is suitable for medical use. In some embodiments, the pharmaceutical composition is suitable for medical use if (a) the G-CSF dimer (e.g., G-CSF-Fc dimer, such as abegrastim alpha) is from about 1 mg / mL to about 100 mg / mL; (b) the buffer (e.g., sodium acetate) is from about 1 mM to about 50 mM; (c) the stabilizer (e.g., EDTA) is from about 0.1 mM to about 20 mM; (d) the tonicity agent (e.g., sorbitol) is from about 1% (w / v) to about 15% (w / v) (e.g., from about 1% (w / v) to about 10% (w / v)); (e) the surfactant (e.g., polysorbate 20) is from about 0.001% (w / v) to about 0.1% (w / v); and (f) the pH of the pharmaceutical composition is from about 4.2 to about 6.2 (e.g., from about 5.0 to about 5.4). In some embodiments, the pharmaceutical composition is suitable for medical use if (a) G-CSF dimer (e.g., G-CSF-Fc dimer, such as abegrastim α) is about 18 mg / mL to about 22 mg / mL (or 20.0±2.0 mg / mL, such as about 20 mg / mL); (b) sodium acetate is about 10 mM; (c) EDTA is about 1 mM; (d) sorbitol is about 5% (w / v); (e) polysorbate 20 is about 0.01% (w / v); and (f) the pH of the pharmaceutical composition is about 5.0 to about 5.4 (or 5.2±0.2, such as about 5.2). 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.

[0149] In some embodiments, the quality control parameters include the effectiveness of the pharmaceutical composition (e.g., before and / or after storage). In some embodiments, for example, by cell proliferation assay, about 70% to about 130% (e.g., about 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120% or 130%) of the effectiveness (e.g., the effectiveness of G-CSF) indicates that the pharmaceutical composition is suitable for medical use. 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, 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. 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, such as measuring tyrosine phosphorylation of JAK kinases or transcription factors), cell proliferation and / or differentiation assays (e.g., using cells expressing G-CSF receptors, such as neutrophils), 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 described in the art; for example, see 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. For example, 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 sera from normal healthy persons and patients with intrafectious and hematological disorders, Exp. Hematol. (1989) 17, 116-119 describes the measurement of G-CSF activity in vitro.For the measurement of G-CSF activity in vivo, see, for example, 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. Other publications describing tests for measuring G-CSF activity are US Pat. No. 6,555,660; 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.

[0150] In some embodiments, the quality control parameter includes 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 any one of 97%, 98%, 99%, or 100%) of G-CSF dimer in a pharmaceutical composition as measured by SEC indicates that the pharmaceutical composition is suitable for medical use. In some embodiments, a pharmaceutical composition comprising at least about 95.5% (e.g., at least about any one of 96%, 97%, 98%, 99%, or 100%) of G-CSF dimer as measured by rCE-SDS indicates that the pharmaceutical composition is suitable for medical use. In some embodiments, a pharmaceutical composition comprising at least about 94% (e.g., at least about any one of 95%, 96%, 97%, 98%, 99%, or 100%) of G-CSF dimer as measured by nrCE-SDS indicates that the pharmaceutical composition is suitable for medical use. In some embodiments, a pharmaceutical composition comprising no more than about 1.5% (e.g., no more than about 1.4%, 1.3%, 1.2%, 1.1%, 1.0%, 0.8%, 0.5%, 0.3%, 0.1% or less) of HMWS as measured by rCE-SDS indicates that the pharmaceutical composition is suitable for medical use. In some embodiments, a pharmaceutical composition comprising no more than about 1.5% (e.g., no more than about 1.4%, 1.3%, 1.2%, 1.1%, 1.0%, 0.8%, 0.5%, 0.3%, 0.1% or less) of HMWS as measured by nrCE-SDS indicates that the pharmaceutical composition is suitable for medical use. In some embodiments, a pharmaceutical composition comprising an acidic region of about 27.2% to about 39.0% (e.g., about 30% to about 40%, such as about 32% to about 37%), a main peak of about 36.5% to about 60.1% (e.g., about 45% to about 55%, such as about 47% to about 55%), and a basic region of about 7.2% to about 30% (e.g., about 10% to about 20%, such as about 11% to 18%) as measured by icIEF indicates that the pharmaceutical composition is suitable for medical use. In some embodiments, a change in the concentration (e.g., G-CSF dimer concentration) of the pharmaceutical composition 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 that the pharmaceutical composition is suitable for medical use. In some embodiments, a concentration of the pharmaceutical composition (e.g., G-CSF dimer concentration) of 20.0 ± 2.0 mg / mL (e.g., by UV spectroscopy scanning) indicates that the pharmaceutical composition is suitable 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, 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.

[0151] In some embodiments, the quality control parameters include viscosity (e.g., before and / or after storage). In some embodiments, if the viscosity of the pharmaceutical composition between about 5°C to about 25°C is about 1 to about 50 mPa-S, such as about 1 to about 45 mPa-S, about 1 to 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 2 mPa-S, it is indicated that the pharmaceutical composition is suitable for medical use. For example, in some embodiments, if the viscosity of the pharmaceutical composition at about 25°C is about 1-2 mPa-S, it is indicated that the pharmaceutical composition is suitable for medical use. In some embodiments, if the pharmaceutical composition is isotonic, it indicates that the pharmaceutical composition is suitable for medical use. In some embodiments, the quality control parameters are measured before storage. In some embodiments, quality control parameters are measured 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.

[0152] In some embodiments, quality control parameters include osmotic pressure molar concentration (for example, before and / or after storage). In some embodiments, if the osmotic pressure molar concentration of the pharmaceutical composition is about 100 to about 500mOsm / kg, for example, about 120 to about 480mOsm / kg, about 140 to about 440mOsm / kg, about 160 to about 420mOsm / kg, about 180 to about 400mOsm / kg, about 200 to about 380mOsm / kg, about 220 to about 360mOsm / kg, about 270 to about 350mOsm / kg / kg or about 268 to about 360mOsm / kg / kg, then it is shown that the pharmaceutical composition is suitable for medical use. In some embodiments, if the osmotic pressure molar concentration of the pharmaceutical composition is about 310mOsm / kg, then it is shown that the pharmaceutical composition is suitable for medical use. 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, 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.

[0153] In some embodiments, the quality control parameters include protein aggregation (e.g., before and / or after storage). In some embodiments, if the percentage of total protein aggregation is less than about 5%, for example, less than about 4%, 3%, 2%, 1%, 0.5%, 0.2%, 0.1% or any one of less, it indicates that the pharmaceutical composition is suitable for medical use. In some embodiments, if the percentage of total protein aggregation measured by SEC is less than about 2% (e.g., less than about 1.5%, 1%, 0.5%, 0.2%, 0.1% or any one of less), it indicates that the pharmaceutical composition is suitable for medical use. 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, 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.

[0154] In some embodiments, the quality control parameters include the percentage and / or amount of HMWS (e.g., before and / or after storage). In some embodiments, if the pharmaceutical composition is stored at 25±2°C for at least about 1 month and contains no more than about 1.5% (e.g., no more than about 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.8%, 0.5%, 0.1% or any one of less, such as no more than about 1%) of HMWS as measured by rCE-SDS, it indicates that the pharmaceutical composition is suitable for medical use. In some embodiments, if the pharmaceutical composition is stored at 25±2°C for at least about 2 months and contains no more than about 1.5% (e.g., no more than about 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.8%, 0.5%, 0.1% or any one of less, such as no more than 1%) of HMWS as measured by rCE-SDS, it indicates that the pharmaceutical composition is suitable for medical use. In some embodiments, if the pharmaceutical composition contains no more than about 1.5% (e.g., no more than 1%) HMWS as measured by rCE SDS after storage at 25±2°C for up to about 3 months, it is indicated that the pharmaceutical composition is suitable for medical use. In some embodiments, if the pharmaceutical composition contains no more than about 1.5% (e.g., no more than about 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.8%, 0.5%, 0.1% or any one of less) HMWS as measured by rCE-SDS after storage at about 2°C to about 8°C for at least about 36 months, it is indicated that the pharmaceutical composition is suitable for medical use. In some embodiments, if the pharmaceutical composition contains no more than about 1.5% HMWS as measured by rCE-SDS after storage at about 2°C to about 8°C for up to about 36 months, it is indicated that the pharmaceutical composition is suitable for medical use. In some embodiments, if the pharmaceutical composition contains no more than about 1.5% (e.g., no more than about 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.8%, 0.5%, 0.1% or less) HMWS by nrCE-SDS after storage at 25±2°C for at least about 1 month, it indicates that the pharmaceutical composition is suitable for medical use. In some embodiments, if the pharmaceutical composition contains no more than about 1.5% (e.g., no more than about 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.8%, 0.5%, 0.1% or less) HMWS by nrCE-SDS after storage at 25±2°C for at least about 2 months, it indicates that the pharmaceutical composition is suitable for medical use. In some embodiments, if the pharmaceutical composition contains no more than about 1.5% HMWS by nrCE-SDS after storage at 25±2°C for up to about 3 months, it indicates that the pharmaceutical composition is suitable for medical use.In some embodiments, if the pharmaceutical composition comprises no more than about 1.5% (e.g., no more than about 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.8%, 0.5%, 0.1%, or any less) of HMWS as measured by nrCE-SDS after storage at about 2°C to about 8°C for at least about 36 months, the pharmaceutical composition is suitable for medical use. In some embodiments, if the pharmaceutical composition comprises no more than about 1.5% of HMWS as measured by nrCE-SDS after storage at about 2°C to about 8°C for up to about 36 months, the pharmaceutical composition is suitable for medical use.

[0155] In some embodiments, the quality control parameters include the percentage and / or amount of low molecular weight substances (LMWS) (e.g., before and / or after storage). In some embodiments, if the pharmaceutical composition comprises no more than about 4% (e.g., no more than about 3.5%, 3%, 2.5%, 2%, 1.5%, 1%, 0.8%, 0.5%, 0.1% or less) of LMWS by rCE-SDS measurement, it is indicated that the pharmaceutical composition is suitable for medical use. In some embodiments, if the pharmaceutical composition comprises no more than about 6% (e.g., no more than about 5.5%, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.5%, 1%, 0.8%, 0.5%, 0.1% or less) of LMWS by nrCE-SDS measurement, it is indicated that the pharmaceutical composition is suitable for medical use. 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, 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.

[0156] In some embodiments, the quality control parameter comprises the percentage and / or amount of G-CSF dimer (e.g., before and / or after storage). In some embodiments, if 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%) of G-CSF dimer, it indicates that the pharmaceutical composition is suitable 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 comprise storage at about 2°C to about 8°C for up to about 36 months. In some embodiments, the storage conditions comprise storage at 25±2°C for up to about 3 months. In some embodiments, if the pharmaceutical composition comprises at least about 95.5% (e.g., at least about any one of 96%, 97%, 98%, 99%, or 100%) of G-CSF dimer as measured by rCE-SDS after storage at least about 1 month at 25±2°C, the pharmaceutical composition is suitable for medical use. In some embodiments, if the pharmaceutical composition comprises at least about 95.5% (e.g., at least about any one of 96%, 97%, 98%, 99%, or 100%) of G-CSF dimer as measured by rCE-SDS after storage at least about 2 months at 25±2°C, the pharmaceutical composition is suitable for medical use. In some embodiments, if the pharmaceutical composition comprises at least about 95.5% of G-CSF dimer as measured by rCE-SDS after storage at 25±2°C for up to about 3 months, the pharmaceutical composition is suitable for medical use. In some embodiments, if the pharmaceutical composition comprises at least about 95.5% (e.g., at least about any one of 96%, 97%, 98%, 99%, or 100%) of G-CSF dimer as measured by rCE-SDS after storage at about 2°C to about 8°C for at least about 36 months, the pharmaceutical composition is suitable for medical use. In some embodiments, if the pharmaceutical composition comprises at least about 95.5% of G-CSF dimer as measured by rCE-SDS after storage at about 2°C to about 8°C for up to about 36 months, the pharmaceutical composition is suitable for medical use. In some embodiments, if the pharmaceutical composition comprises at least about 94% (e.g., at least about any one of 95%, 96%, 97%, 98%, 99%, or 100%) of G-CSF dimer as measured by nrCE-SDS after storage at 25±2°C for at least about 1 month, the pharmaceutical composition is suitable for medical use. In some embodiments, a pharmaceutical composition is suitable for medical use if it comprises 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.In some embodiments, if the pharmaceutical composition comprises at least about 94% G-CSF dimer as measured by nrCE-SDS after storage at 25±2°C for up to about 3 months, the pharmaceutical composition is suitable for medical use. In some embodiments, if the pharmaceutical composition comprises 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 about 2°C to about 8°C for at least about 36 months, the pharmaceutical composition is suitable for medical use. In some embodiments, if the pharmaceutical composition comprises at least about 94% G-CSF dimer as measured by nrCE-SDS after storage at about 2°C to about 8°C for up to about 36 months, the pharmaceutical composition is suitable for medical use.

[0157] In some embodiments, the quality control parameter comprises the percentage of G-CSF dimer as measured by SEC (e.g., before and / or after storage). In some embodiments, if the pharmaceutical composition comprises at least about 96% (e.g., at least about any one of 97%, 98%, 99%, or 100%) of G-CSF dimer as measured by SEC, it indicates that the pharmaceutical composition is suitable for medical use. In some embodiments, when measured by SE-HPLC, if the pharmaceutical composition comprises a main peak of at least about 96% (e.g., at least about any one of 97%, 98%, 99%, or 100%) relative to the total area of ​​all peaks, it indicates that the pharmaceutical composition is suitable 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 comprise storage at about 2°C to about 8°C for up to about 36 months. In some embodiments, the storage conditions comprise storage at 25±2°C for up to about 3 months. In some embodiments, a pharmaceutical composition is suitable for medical use if it comprises at least about 96% (e.g., at least about any of 97%, 98%, 99%, or 100%) of G-CSF dimer as measured by SE-HPLC after storage at least about 1 month at 25±2°C. In some embodiments, a pharmaceutical composition is suitable for medical use if it comprises at least about 96% (e.g., at least about any of 97%, 98%, 99%, or 100%) of G-CSF dimer as measured by SE-HPLC after storage at least about 2 months at 25±2°C. In some embodiments, a pharmaceutical composition is suitable for medical use if it comprises at least about 96% of G-CSF dimer as measured by SE-HPLC after storage at 25±2°C for up to about 3 months. In some embodiments, a pharmaceutical composition is suitable for medical use if it comprises at least about 96% (e.g., at least any of about 97%, 98%, 99%, or 100%) of G-CSF dimer as measured by SE-HPLC after storage at about 2°C to about 8°C for at least about 36 months. In some embodiments, a pharmaceutical composition is suitable for medical use if it comprises at least about 96% of G-CSF dimer as measured by SE-HPLC after storage at about 2°C to about 8°C for up to about 36 months. In some embodiments, a pharmaceutical composition is suitable for medical use if it comprises at least about 85% (e.g., at least any of about 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of a main peak 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, when measured by SE-HPLC after storage at 25±2°C for at least about 2 months, if the pharmaceutical composition comprises a main peak of at least about 85% (e.g., at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100%) relative to the total area of ​​all peaks, it is indicated that the pharmaceutical composition is suitable for medical use. In some embodiments, when measured by SE-HPLC after storage at 25±2°C for up to about 3 months, if the pharmaceutical composition comprises a main peak of at least about 85% relative to the total area of ​​all peaks, it is indicated that the pharmaceutical composition is suitable for medical use. In some embodiments, when measured by SE-HPLC after storage at about 2°C to about 8°C for at least about 36 months, if the pharmaceutical composition comprises a main peak of at least about 85% (e.g., at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100%) relative to the total area of ​​all peaks, it is indicated that the pharmaceutical composition is suitable for medical use. In some embodiments, a pharmaceutical composition is suitable for medical use if it comprises a major peak of at least about 85% relative to the total area of ​​all peaks when measured by SE-HPLC after storage at about 2°C to about 8°C for up to about 36 months.

[0158] In some embodiments, the quality control parameters include the percentage of dimers measured by SEC (e.g., before and / or after storage). In some embodiments, when measured by SE-HPLC, if the pharmaceutical composition contains dimers less than about 5% (e.g., less than about 4%, 3%, 2%, 1.5%, 1%, 0.5%, 0.1% or any one of less, such as less than about 2.5%) relative to the total area of ​​all peaks, it is indicated that the pharmaceutical composition is suitable for medical use. 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, 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. In some embodiments, when measured by SE-HPLC after storage at about 25±2°C for at least about 1 month, if the pharmaceutical composition comprises less than about 5% (e.g., less than about 4%, 3%, 2%, 1.5%, 1%, 0.5%, 0.1% or any one of less, such as less than about 2.5%) of dimer relative to the total area of ​​all peaks, then the pharmaceutical composition is suitable for medical use. In some embodiments, when measured by SE-HPLC after storage at 25±2°C for at least about 2 months, if the pharmaceutical composition comprises less than about 5% (e.g., less than about 4%, 3%, 2%, 1.5%, 1%, 0.5%, 0.1% or any one of less, such as less than about 2.5%) of dimer relative to the total area of ​​all peaks, then the pharmaceutical composition is suitable for medical use. In some embodiments, when measured by SE-HPLC after storage for up to about 3 months at 25±2°C, if the pharmaceutical composition comprises less than about 5% (e.g., less than about 2.5%) dimer relative to the total area of ​​all peaks, it indicates that the pharmaceutical composition is suitable for medical use. In some embodiments, when measured by SE-HPLC after storage for at least about 36 months at about 2°C to about 8°C, if the pharmaceutical composition comprises less than about 5% (e.g., less than about 4%, 3%, 2%, 1.5%, 1%, 0.5%, 0.1% or any one of less, such as less than about 2.5%) dimer relative to the total area of ​​all peaks, it indicates that the pharmaceutical composition is suitable for medical use. In some embodiments, when measured by SE-HPLC after storage for up to about 36 months at about 2°C to about 8°C, if the pharmaceutical composition comprises less than about 5% (e.g., less than about 2.5%) dimer relative to the total area of ​​all peaks, it indicates that the pharmaceutical composition is suitable for medical use.

[0159] In some embodiments, the quality control parameters include peak values ​​measured by icIEF (e.g., before and / or after storage). In some embodiments, after storage at 25±2°C for at least about 1 month, if the pharmaceutical composition (e.g., liquid pharmaceutical composition) comprises about 27.2% to about 39.0% (e.g., about 32% to about 37%) of the acidic region, about 36.5% to about 60.1% (e.g., about 47% to about 55%) of the main peak, and about 7.2% to about 30% (e.g., about 11% to about 18%) of the basic region, as measured by icIEF, the pharmaceutical composition is suitable for medical use. In some embodiments, after storage at 25±2°C for at least about 2 months, if the pharmaceutical composition (e.g., a liquid pharmaceutical composition) comprises about 27.2% to about 39.0% (e.g., about 32% to about 37%) of an acidic region, about 36.5% to about 60.1% (e.g., about 47% to about 55%) of a main peak, and about 7.2% to about 30% (e.g., about 11% to about 18%) of a basic region as measured by icIEF, the pharmaceutical composition is suitable for medical use. In some embodiments, after storage at 25±2°C for up to about 3 months, if the pharmaceutical composition (e.g., a liquid pharmaceutical composition) comprises about 27.2% to about 39.0% of an acidic region, about 36.5% to about 60.1% of a main peak, and about 7.2% to about 30% of a basic region as measured by icIEF, the pharmaceutical composition is suitable for medical use. In some embodiments, after storage at about 2°C to about 8°C for at least about 36 months, the liquid pharmaceutical composition comprises about 27.2% to about 39.0% (e.g., about 32% to about 37%) of the acidic region, about 36.5% to about 60.1% (e.g., about 47% to about 55%) of the main peak, and about 7.2% to about 30% (e.g., about 11% to about 18%) of the basic region as measured by icIEF, indicating that the pharmaceutical composition is suitable for medical use. In some embodiments, after storage at about 2°C to about 8°C for up to about 36 months, the liquid pharmaceutical composition comprises about 27.2% to about 39.0% of the acidic region, about 36.5% to about 60.1% of the main peak, and about 7.2% to about 30% of the basic region as measured by icIEF, indicating that the pharmaceutical composition is suitable for medical use.

[0160] In some embodiments, the method comprises evaluating two or more of the above-mentioned quality control parameters, including, for example, any one of at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more of the above-mentioned quality control parameters.

[0161] Also provided are methods for releasing a batch (e.g., a commercial batch) of a pharmaceutical composition described herein, the method comprising: using a sample of the commercial batch to evaluate the suitability of the pharmaceutical composition for medical use in an individual (e.g., a human individual) according to any of the methods described herein (e.g., by evaluating whether one or more quality control parameters described herein are within a quality control parameter threshold), and if the sampled pharmaceutical composition is suitable for medical use, releasing the commercial batch.

[0162] Also provided are methods of processing a sample of any pharmaceutical composition described herein to verify whether the sample is suitable for medical use in an individual (e.g., a human individual), the method comprising: obtaining a sample from a commercial batch; and using the sample from the commercial batch according to any method described herein to assess the suitability of the sampled pharmaceutical composition for medical use in an individual (e.g., a human individual), for example by assessing whether one or more quality control parameters described herein are within a quality control parameter threshold.

[0163] Also provided are commercial batches of any pharmaceutical composition described herein, wherein the commercial batch is optionally evaluated, validated, released, or processed according to any method described herein.

[0164] IV, G-CSF molecule

[0165] Human G-CSF is a glycoprotein with 204 amino acids and a 30 amino acid signal peptide. The molecular weight of the mature G-CSF protein is 18-20 kDa, composed of 174 amino acids, without a signal peptide, and is secreted from cells. The human cells primarily responsible for this secretion are monocytes, fibroblasts, and endothelial cells. G-CSF has three major biological functions, namely: 1) acting on bone marrow precursors and stem cells to drive the differentiation, development, and maturation of neutrophils; 2) activating mature neutrophils to participate in immune responses; 3) acting with other hematopoietic growth factors such as stem cell factor, Flt-3 ligand, and GM-CSF to mobilize hematopoietic stem cells.

[0166] The G-CSF receptor (G-CSFR) has been shown to be present in bone marrow hematopoietic stem cells Sca+Lin-Th1low, precursor cells CD34+, committed granulocyte precursor cells and mature neutrophils. G-CSFR is a specific receptor with high affinity for G-CSF and 812 amino acids. Tamada et al. obtained the crystal structure of the G-CSF:G-CSFR complex, and showed that the stoichiometric ratio of the G-CSF:G-SFR complex was 2:2 by 2.8 angstrom diffraction analysis (PNAS, 2008, Vol. 103: 3135-3140). In other words, in each complex, when two G-CSF receptor complexes are close, each G-CSF will bind to one receptor chain to form a G-CSF receptor complex, and this interaction will form a 2:2 dimer. In this case, the carboxyl terminus of the G-CSF receptor is then able to activate the downstream signaling molecule JAKs (Janus tyrosine kinase, mainly JAK2). Thus, JAK2 activates STAT3 to turn on transcriptional genes that are critical for neutrophil differentiation, proliferation, and activation.

[0167] In 2003, Schhabitz WR et al. reported that in the study of ischemic animal models, recombinant human G-CSF (rhG-CSF) was shown to have a protective function on nerve cells (Storke, 2003, 34; 745-751). Later in 2006, Shyu et al. reported that rhG-CSF showed clinical efficacy in the treatment of acute stroke patients, where patients took rhG-CSF every day for five consecutive days (CMAJ, 2006174: 927-933). The in vivo half-life of rat G-CSF after subcutaneous administration is about 2 hours, while the half-life of human G-CSF after subcutaneous administration is only 3.5 hours. Therefore, it is necessary to administer drugs or intravenous infusions to patients in need every day, which will affect the quality of life of patients.

[0168] Pegfilgrastim, also known as SD-01 and PEG-rmetHuG-CSF, is a G-CSF drug that has been developed to reduce the severity and duration of severe neutropenia associated with the administration of myelosuppressive anticancer drugs or radiotherapy, as well as complications of neutropenia. Pegylated filgrastim consists of recombinant methoxy human G-CSF (r-metHuG-CSF) covalently linked to monomethoxy polyethylene 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. It is believed that the conjugation of mPEG to the N-terminal methionine of r-metHuG-CSF increases the serum half-life of r-metHuG-CSF (about 15-80 hours), thereby reducing the frequency of administration required to maintain therapeutic neutrophil counts.

[0169] Eflapegrastim, also known as SPI-2012, HM10460A and 17, 65 SG-CSF, a long-acting G-CSF drug, has been developed to reduce the severity and duration of severe neutropenia associated with the use of myelosuppressive anticancer drugs or radiotherapy, as well as complications of neutropenia. Efapiglastin consists of a recombinant human G-CSF analog (ef-G-CSF) and a recombinant fragment of the Fc region of human IgG4 connected by a bifunctional polyethylene glycol linker. ef-G-CSF differs from human G-CSF (SEQ ID NO: 1) at positions 17 and 65, where positions 17 and 65 are replaced by serine. It is believed that the Fc region of human IgG4 increases the serum half-life of ef-G-CSF to about 36.4 hours (range: about 16.1 to about 115 hours).

[0170] Both pegfilgrastim and efavirenzostatin have the G-CSF molecule in monomeric form.

[0171] G-CSF-based drugs, such as filgrastim and its biosimilar Filgrastimaafi Filgrastim sndz Filgrastim ayow For the treatment of neutropenia in patients receiving chemotherapy drugs. However, these filgrastim or similar drugs need to be administered daily because of their short half-life (3.5 hours). Other G-CSF drugs, such as pegfilgrastim (e.g. ) and efavirenz (eg, ROLVEDONTM ), which can be administered once per chemotherapy cycle due to its extended half-life of 30-50 hours through pegylation technology or fusion protein strategy. The recommended dosing schedule for both efavirenz (HM10460A) and efavirenz (Effexor) is to be administered on the second day after cytotoxic chemotherapy.

[0172] V. G-CSF dimer

[0173] 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.

[0174] As used herein, the term "G-CSF dimer" refers to a protein comprising (or consisting essentially of): 2 units of a G-CSF molecule (e.g., two units of any G-CSF monomer described herein, or comprising 2 units of any monomer subunit of any G-CSF described herein). In one non-limiting example, a G-CSF dimer may comprise (or consist essentially of, or consist of): two G-CSF monomers directly linked to each other, or two G-CSF monomers linked together by a linking moiety such as 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 may comprise two monomer subunits, each of which comprises a G-CSF monomer linked to a carrier protein (e.g., albumin or an Fc domain). Other examples of G-CSF dimers useful 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. 20130165637, the entire contents of which are incorporated herein by reference.

[0175] As used herein, the term "G-CSF monomer" refers to one unit of a G-CSF protein or molecule. The terms "G-CSF," "G-CSF molecule," and "G-CSF protein" are used interchangeably herein.

[0176] The G-CSF monomer used in the G-CSF dimer can be derived from the G-CSF molecule of any organism, such as humans or non-human animals, including but not limited to mammals, such as primates (such as cynomolgus monkeys, rhesus monkeys), cattle, pigs, horses, sheep, goats, rodents, cats and dogs. The G-CSF monomer can be wild-type G-CSF or mutant G-CSF, such as mutant G-CSF that can produce 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 that can produce 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.

[0177] 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., CHO cell). In some embodiments, the G-CSF dimer comprises (or consists essentially of): two G-CSF (e.g., hG-CSF) monomers, for example, two G-CSF monomers connected to each other by a linker (e.g., a peptide linker). In some embodiments, the two G-CSF monomers forming the G-CSF dimer are identical (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.

[0178] In some embodiments, the 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 identical (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.

[0179] In some embodiments, the G-CSF dimer comprises formula (I):

[0180] M1-L-M2(I)

[0181] wherein M1 is a first monomer of G-CSF; M2 is a second monomer of G-CSF; and L is a linker connecting the first monomer and the second monomer and disposed therebetween. In some embodiments, the linker L is selected from: i) a short peptide comprising about 3 to about 50 amino acids; and ii) a polypeptide of formula (II):

[0182] -ZYZ-(II)

[0183] Wherein Y is a carrier protein (e.g., albumin, Fc domain); Z is empty or a short peptide comprising about 1 to about 30 amino acids; "-" is a chemical bond or a covalent bond. Any suitable linker or short peptide can be used herein, which can provide flexibility between two G-CSF monomers or between a G-CSF monomer and a carrier protein, and / or ensure the binding of each G-CSF monomer to its receptor. In some embodiments, the linker L or short peptide Z comprises the amino acid sequence of any one of SEQ ID NOs: 12-31.

[0184] The carrier protein described herein can be any protein suitable for linking two G-CSF monomers to form a G-CSF dimer, including but not limited to an Fc fragment of an immunoglobulin (e.g., human IgG1, IgG2, IgG3, IgG4) or an albumin (e.g., human serum albumin). When a carrier protein is formed by linking two protein subunits (e.g., by a disulfide bond, a peptide bond, or a chemical bond), each protein subunit is referred to as a dimerization domain. In some embodiments, the carrier protein is formed by linking two dimerization domains (e.g., two Fc fragments of IgG) via 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, the carrier protein is formed by linking a first Fc fragment and a different second Fc fragment via one or more disulfide bonds.

[0185] In some embodiments, the G-CSF dimer comprises (or consists essentially of, or consists of): two monomer subunits, wherein each monomer subunit comprises (or consists essentially of, or consists of): a G-CSF monomer (e.g., an hG-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".

[0186] 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% (e.g., at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater) sequence identity 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.

[0187] In some embodiments, within each monomer subunit, the G-CSF monomer is directly linked to the dimerization domain.

[0188] In some embodiments, within each monomer subunit, the G-CSF monomer is connected to the dimerization domain by an optional linker. In some embodiments, the linker is about 6 to about 30 amino acids in length, such as about any of 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 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 one of SEQ ID NOs: 12-31. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 12.

[0189] The linker between two G-CSF monomers, or between a G-CSF monomer and a carrier protein or dimerization domain, can be empty (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) and the second linker connecting the second G-CSF monomer and the protein (or the second dimerization domain) are the same. In some embodiments, the first linker connecting the first G-CSF monomer and the carrier protein (or the first dimerization domain) and the second linker connecting the second G-CSF monomer and the protein (or the second dimerization domain) are different. In general, the linker does not affect or significantly affect the correct folding and conformation formed by the configuration of the two G-CSF monomers.

[0190] The joint can be a peptide joint of any length. In some embodiments, the peptide joint is about 1 amino acid (aa) to about 10 aa, about 2 aa to about 15 aa, about 5 aa to about 8 aa, about 1 aa to about 20 aa, about 21 aa to about 30 aa, about 1 aa to about 30 aa, about 10 aa to about 30 aa, about 3 aa to about 50 aa, or about 6 aa to about 30 aa. In some embodiments, the length of the peptide joint 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 some embodiments, the length of the peptide joint is about 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids. In some embodiments, the length of the peptide linker is about 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids. For example, in some embodiments, the length of the linker is about 3 to about 50 amino acids. In some embodiments, the length of the linker is about 6 to about 30 amino acids.

[0191] The peptide linker can have a naturally occurring sequence or a non-naturally occurring sequence. For example, a sequence derived from only the hinge region of a heavy chain antibody can be used as a linker. For example, see WO1996 / 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 (including, for example, (GS) n (SEQ ID NO: 19), (GSGGS) n (SEQ ID NO: 20) or (GGGGGGS) n(SEQ ID NO: 22), wherein 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 therefore may be able to be used as neutral tethers between components. Glycine even obtains significantly more phi-psi space than alanine and is much 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 one of SEQ ID NOs: 12 and 14-31. In some embodiments, the linker comprises (or consists essentially of, or consists of): the sequence of SEQ ID NO: 12. In some embodiments, the linker (e.g., comprising SEQ ID NO: 12) also comprises the sequence of SEQ ID NO: 13. One of ordinary skill in the art will recognize that the design of a G-CSF dimer can include a fully or partially flexible linker, such that the linker can include a flexible linker portion and one or more portions that impart a less flexible structure to provide the desired G-CSF dimer structure and function.

[0192] In some embodiments, the linker between a G-CSF monomer and a carrier protein (eg, a dimerization domain) or between two G-CSF monomers is a stable linker (not cleavable by proteases, particularly MMPs).

[0193] Other linker considerations include the effect on the physical or pharmacokinetic properties of the resulting G-CSF dimer, such as solubility, lipophilicity, hydrophilicity, hydrophobicity, stability (more or less stable as well as planned degradation), rigidity, flexibility, immunogenicity, modulation of G-CSF / G-CSF receptor binding, ability to be incorporated into micelles or liposomes, etc.

[0194] In some embodiments, the dimerization domain (e.g., Fc fragment) within each monomer subunit comprises at least two cysteines capable of forming intermolecular disulfide bonds. In some embodiments, there are about 2 to about 4 disulfide bonds between two dimerization domains (e.g., Fc fragments). In some embodiments, the dimerization domain includes a leucine zipper. In some embodiments, the dimerization domain includes at least a portion of the Fc fragment. In some embodiments, the Fc fragment includes CH2 and CH3 domains. In some embodiments, the dimerization domain is derived from the Fc fragment of any one of IgA, IgD, IgE, IgG, and IgM and its subtype. 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 dimerization domain is derived from the Fc fragment of human IgG2. In some embodiments, the dimerization domain is derived from the Fc fragment of human IgG1. In some embodiments, the dimerization domain is derived from the 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, to reduce or eliminate effector functions, such as reducing antibody-dependent cellular toxicity (ADCC) or reducing binding to FcγR. In some embodiments, the Fc fragment comprises L234A and L235A ("LALA") replacements (EU numbering). In some embodiments, the dimerization domain is an IgG2 Fc fragment comprising a P331S replacement according to EU numbering. In some embodiments, the IgG2 Fc fragment comprises a sequence of SEQ ID NO:2 or 3. In some embodiments, the dimerization domain is an IgG1 Fc fragment comprising L234A, L235A and P331S replacements according to EU numbering. In some embodiments, the IgG1 Fc fragment comprises a sequence of SEQ ID NO:4. In some embodiments, the dimerization domain is an IgG4 Fc fragment comprising S228P, L234A and L235A replacements according to EU numbering. In some embodiments, the IgG4 Fc fragment comprises a 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-terminal truncated Fc fragment, such as a truncated Fc fragment with less N-terminal cysteine ​​to reduce disulfide bond mispairing during dimerization. In some embodiments, the Fc fragment is truncated at the N-terminus, such as lacking the first 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids of the 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.

[0195] In some embodiments, each monomer subunit comprises an Fc fragment of human IgG2 or a variant thereof, the variant comprising up to 5, 4, 3, 2 or 1 amino acid substitutions. In some embodiments, each monomer subunit comprises an Fc fragment of human IgG1 or a variant thereof, the variant comprising up to 5, 4, 3, 2 or 1 amino acid substitutions. In some embodiments, each monomer subunit comprises an Fc fragment of human IgG4 or a variant thereof, the variant comprising up to 5, 4, 3, 2 or 1 amino acid substitutions. In some embodiments, each monomer subunit comprises an Fc fragment having at least about 95% (e.g., at least about 96%, 97%, 98%, 99% or more) sequence identity with SEQ ID NO: 2-5. In some embodiments, the Fc fragment comprises (or consists essentially of, or consists of): the sequence of any one of SEQ ID NO: 2-5. In some embodiments, the Fc fragment comprises (or consists essentially of, or consists of): the sequence of SEQ ID NO: 2.

[0196] In some embodiments, the G-CSF dimer comprises two monomer subunits, wherein: i) each monomer 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 monomer 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 monomer 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 monomer 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 monomer subunits, wherein each monomer 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 linked to the Fc fragment via a linker (eg, a linker comprising the amino acid sequence of SEQ ID NO: 12).

[0197] In some embodiments, the G-CSF monomer is C-terminal to the dimerization domain within each monomer subunit. In some embodiments, the G-CSF monomer is N-terminal to the dimerization domain within each monomer subunit.

[0198] In some embodiments, each monomer subunit comprises (or consists essentially of, or consists of): an amino acid sequence of SEQ ID NO: 6, or a variant thereof, the variant having at least about 90% (e.g., at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater) sequence identity with the amino acid sequence of SEQ ID NO: 6. In some embodiments, each monomer subunit comprises (or consists essentially of, or consists of): an amino acid sequence of SEQ ID NO: 7, or a variant thereof, the variant having at least about 90% (e.g., at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater) sequence identity with the amino acid sequence of SEQ ID NO: 6. In some embodiments, each monomer subunit comprises (or consists essentially of, or consists of): an amino acid sequence of SEQ ID NO: 8, or a variant thereof, a variant having at least about 90% (e.g., at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater) sequence identity with the amino acid sequence of SEQ ID NO: 8. In some embodiments, each monomer subunit comprises (or consists essentially of, or consists of): an amino acid sequence of SEQ ID NO: 9, or a variant thereof, a variant having at least about 90% (e.g., at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater) sequence identity with the amino acid sequence of SEQ ID NO: 8. In some embodiments, each monomer subunit comprises (or consists essentially of, or consists of): an amino acid sequence of SEQ ID NO:10, or a variant thereof, said variant having at least about 90% (e.g., at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater) sequence identity with the amino acid sequence of SEQ ID NO:10.

[0199] In some embodiments, each monomer subunit comprises (or consists essentially of, or consists of): an amino acid sequence of any one of SEQ ID NOs: 6-10. In some embodiments, each monomer subunit comprises (or consists essentially of, or consists of): an amino acid sequence of SEQ ID NO: 6.

[0200] Thus, in some embodiments, the G-CSF dimer comprises two monomer subunits, each monomer subunit comprising (or consisting essentially of, or consisting of) the amino acid sequence of SEQ ID NO: 6. Such a G-CSF dimer (or G-CSF-Fc dimer) is also referred to herein as "F-627" or "Abegrastim alfa". In some embodiments, each monomer 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.

[0201] 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-terminus or C-terminus of one or both monomer subunits of the G-CSF dimer (e.g., a G-CSF monomer or a G-CSF-Fc monomer subunit). In some embodiments, such additional amino acid sequence is beneficial for expression (e.g., a signal peptide), purification (e.g., a 6×His sequence, a cleavage site for the α-factor signal peptide (Glu-Lys-Arg) of Saccharomyces cerevisiae), or for enhancing the biological activity of the G-CSF dimer.

[0202] In some embodiments, the G-CSF dimer is abegrastim alfa. Abegrastim alfa is a long-acting human G-CSF and human IgG2 Fc fragment fusion protein that exists as a dimer composed of two G-CSF-Fc monomer subunits that are covalently linked by a disulfide bond formed between the Fc fragments of the molecule. Each G-CSF-Fc monomer subunit comprises (or consists essentially of, or consists of): the amino acid sequence of SEQ ID NO: 6. When numbering from the N-terminus of the entire polypeptide chain of the G-CSF-Fc monomer subunit, the hIgG2 Fc fragment has a P297S substitution, which corresponds to a P331S substitution located in the CH2 domain according to EU numbering. The purpose of the P331S substitution is to reduce the complement-dependent cytotoxicity function (CDC) mediated by the Fc region of human IgG2. Abegrastim alfa is a glycosylated protein produced by Chinese hamster ovary cells using serum-free medium. Each G-CSF-Fc monomer subunit of abegrastim alfa has an N-linked glycosylation site at N263 when 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 an O-linked glycosylation site at T133 in the human G-CSF monomer. Without wishing to be bound by theory, it is believed that the IgG2-Fc fragment increases the serum half-life of human G-CSF.

[0203] By specifically binding to the G-CSF receptor, abegrastim alfa stimulates the survival, proliferation, differentiation, and function of neutrophil precursors and mature neutrophils. Abegrastim alfa was developed to reduce the incidence of infections as manifested by febrile neutropenia in patients with non-myeloid malignancies receiving myelosuppressive anticancer drugs associated with a clinically significant incidence of febrile neutropenia (abegrastim alfa was administered after (e.g., 24 hours or 48 hours) myelosuppressive anticancer drug treatment).

[0204] a) Substitutions, insertions, deletions and variations

[0205] In some embodiments, G-CSF molecules (e.g., G-CSF dimers) having one or more amino acid variations (e.g., substitutions) are provided. Conservative substitutions are shown in Table 1 under the heading "Preferred Substitutions". More substantial variations are provided 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 a G-CSF molecule (e.g., G-CSF dimer) (e.g., substitutions within one or more of a G-CSF monomer, linker, dimerization domain (e.g., Fc fragment)) and the products screened for a desired activity, such as retained / improved receptor binding, reduced immunogenicity, or improved ADCC or complement dependent cytotoxicity (CDC).

[0206] Table 1. Amino acid substitutions

[0207] Original residue Exemplary Replacements Preferred replacement Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) G1n; His; Asp, Lys; Arg G1n Asp(D) Glu; Asn Glu Cys(C) Ser; Ala Ser Gln(Q) Asn;Glu Asn Glu(E) Asp; Gln Asp Gly(G) Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe; Norleucine Leu Leu(L) Norleucine; Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; 11e Leu Phe(F) Trp;Leu;Val;I1e;Ala;Tyr Tyr Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Val; Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Ala; norleucine Leu

[0208] Amino acids can be grouped according to common side chain properties: (1) Hydrophobic: norleucine, Met (methionine), Ala (alanine), Val (valine), Leu (leucine), Ile (isoleucine); (2) Neutral hydrophilic: Cys (cysteine), Ser (serine), Thr (threonine), Asn (aspartic acid), Gln (glutamine); (3) Acidic: aspartic acid (Asp), glutamic acid (Glu); (4) Basic: His (histidine), Lys (lysine), Arg (arginine); (5) Residues affecting chain orientation: Gly (glycine), Pro (proline); (6) Aromatic: Trp (tryptophan), Tyr (tyrosine), Phe (phenylalanine).

[0209] Non-conservative substitutions will entail exchanging a member of one of these classes for another.

[0210] Alterations (eg, substitutions) may be made, for example, to increase affinity for G-CSF.

[0211] In some embodiments, substitutions, insertions and / or deletions may occur within a G-CSF molecule (e.g., a G-CSF dimer) as long as these changes do not significantly reduce the ability of the G-CSF molecule to bind to a G-CSF receptor. For example, conservative substitutions (e.g., conservative substitutions provided herein) that do not significantly reduce binding affinity may be made.

[0212] A useful method for identifying residues or regions of G-CSF or variants thereof that may be targeted for mutagenesis is called "alanine scanning mutagenesis" as described by Cunningham and Wells (1989) Science, 244: 1081-1085. In this method, residues or groups of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) are identified and replaced with neutral or negatively charged amino acids (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 substitutions. Alternatively or additionally, a crystal structure of the receptor-ligand complex can be prepared to identify contact points between G-CSF and the receptor. Such contact residues and adjacent residues can be targeted or eliminated as candidates for substitution. The variants can be screened to determine whether they contain the desired properties.

[0213] Amino acid sequence insertions include amino and / or carboxyl terminal fusions ranging in length from one residue to polypeptides containing more than one hundred residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include fusion of the N- or C-terminus of a G-CSF molecule to a polypeptide, thereby increasing the half-life of the G-CSF molecule in serum.

[0214] b) Glycosylation variants

[0215] In some embodiments, each G-CSF-Fc monomer subunit in the G-CSF dimer has one N-linked glycosylation site at N297 in the CH2 domain according to EU numbering. In some embodiments, each G-CSF-Fc monomer subunit in the G-CSF dimer has one O-linked glycosylation site at T133 in the hG-CSF monomer relative to SEQ ID NO: 1.

[0216] In some embodiments, the G-CSF molecule (G-CSF dimer) is altered to increase or decrease the extent of protein glycosylation. Addition or deletion of glycosylation sites (e.g., addition or deletion to Fc) can be conveniently accomplished by altering the amino acid sequence to create or delete one or more glycosylation sites.

[0217] When the G-CSF dimer contains an Fc region, the carbohydrate attached to it may vary. Native antibodies produced by mammalian cells typically contain branched biantennary oligosaccharides that are usually attached to the C of the Fc region via N-linkages. H 2 domain. See, e.g., Wright et al., TIBTECH 15:26-32 (1997). Oligosaccharides may include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to the GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, the oligosaccharides in the Fc region may be modified to produce Fc variants with certain improved properties.

[0218] In some embodiments, the 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 may be from about 1% to about 80%, from about 1% to about 65%, from about 5% to about 65%, or from about 20% to about 40%. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at N297 relative to the sum of all sugar structures (e.g., complexes, hybrids, and high mannose structures) attached to N297, as measured, for example, by MALDI-TOF mass spectrometry as described in WO 2008 / 077546. N297 refers to the asparagine residue located at about position 297 of the Fc region (EU numbering of Fc region residues); however, due to minor sequence variations in the Fc domain, N297 may also be located about ±3 amino acids upstream or downstream of position 297, i.e., between position 294 and position 300. Such fucosylation variants may have improved ADCC function. For example, see US Patent Publication Nos. US2003 / 0157108 (Presta, L.); US2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications related to "defucosylated" or "fucose-deficient" proteins include: US 2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; US2003 / 0115614; US2002 / 0164328; US2004 / 0093621; US2004 / 0132140; US2004 / 0110704; US2004 / 0110282; US2004 / 0109865; WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO 2005 / 035778; WO2005 / 053742; WO2002 / 031140; Okazaki et al., Journal of 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 lacking protein fucosylation (Ripka et al., Arch. Biochem. Biophys. 249: 533-545 (1986); U.S. Patent Application No. US2003 / 0157108 A1, Presta, L; and WO 2004 / 056312 A1, Adams et al., particularly in Example 11), and knockout cell lines, such as α-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane Ohnuki et al., Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotech. Bioeng., 94 (4): 680-688 (2006); and WO2003 / 085107).

[0219] In some embodiments, the G-CSF molecule (e.g., a G-CSF dimer, such as a G-CSF-Fc dimer) cleaves an oligosaccharide in two, for example, wherein a biantennary oligosaccharide attached to the Fc region is cleaved in two by GlcNAc. Such variants may have reduced fucosylation and / or improved ADCC function.

[0220] c) Fc region variants

[0221] In some embodiments, the Fc fragment of the G-CSF-Fc dimer has reduced effector function, e.g., at least about any of 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% or more effector function, as compared to a corresponding wild-type Fc fragment, as measured by antibody-dependent cellular cytotoxicity (ADCC) levels.

[0222] In some embodiments, one or more amino acid modifications may be introduced into the Fc fragment of the G-CSF-Fc dimer, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence comprising an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0223] In some embodiments, the Fc fragment possesses some but not all effector functions, making it an ideal candidate for applications where the in vivo half-life of the G-CSF-Fc dimer is important but certain effector functions (such as complement and ADCC) are unnecessary or detrimental. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduction / depletion of CDC and / or ADCC activity. For example, an Fc receptor (FcR) binding assay can be performed to ensure that the G-CSF-Fc dimer lacks FcγR binding (and therefore may lack ADCC activity) but retains FcRn binding ability. The primary cells used to mediate ADCC, i.e., natural killer (NK) cells, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIIII.

[0224] The expression of FcR 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 for evaluating ADCC activity of molecules of interest are described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom 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 assays (e.g., see ACTI of flow cytometry) can be used. TM Nonradioactive cytotoxicity assays (CellTechnology, Inc. Mountain View, CA; and Cytox Non-radioactive cytotoxicity assay (Promega, Madison, WI). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and NK cells. Alternatively or additionally, ADCC activity of the molecule of interest can be assessed in vivo, for example, in an animal model as disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). A C1q binding assay can also be performed to confirm that the G-CSF-Fc dimer cannot bind to C1q and therefore lacks CDC activity. See, for example, C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay can be performed (e.g., see 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)). FcRn binding and in vivo clearance / half-life assays can also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18 (12): 1759-1769 (2006)).

[0225] Fc domain variants with reduced effector function include variants in which one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 are replaced (U.S. Patent No. 6,737,056). Such Fc mutants include Fc mutants in which two or more of amino acid positions 265, 269, 270, 297, and 327 are replaced, including the so-called "DANA" Fc mutants in which residues 265 and 297 are replaced with 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.

[0226] Certain Fc variants with improved or reduced binding to FcRs have been described. See, for example, U.S. Patent No. 6,737,056; WO 2004 / 056312 and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001).

[0227] In some embodiments, the Fc region is altered resulting in altered (ie, improved or decreased) CIq binding and / or CDC, e.g., as described in U.S. Patent No. 6,194,551, WO 99 / 51642, and Idusogie et al., J. Immunol. 164:4178-4184 (2000).

[0228] In some embodiments, the G-CSF-Fc dimer comprises a variant Fc region comprising one or more amino acid substitutions that alter half-life and / or alter binding to the neonatal Fc receptor (FcRn). US2005 / 0014934A1 (Hinton et al.) describes Fc domains with increased half-life and improved binding to FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)). Those Fc domains having one or more substitutions alter binding of the Fc region to FcRn, such as substitution of residue 434 of the Fc region (U.S. Pat. No. 7,371,826).

[0229] 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 for other examples of Fc region variants.

[0230] In some embodiments, it may be desirable to create a cysteine ​​engineered G-CSF dimer moiety in which one or more residues of the G-CSF dimer are replaced with cysteine ​​residues. In particular embodiments, the replaced residues occur at accessible sites of the G-CSF dimer. By replacing these residues with cysteine, reactive sulfhydryl groups are thereby positioned at accessible sites of the G-CSF dimer and can be used to conjugate the G-CSF dimer to other moieties. In some embodiments, any one or more of the following residues may be replaced with cysteine: A118 (EU numbering) in the hinge region and S400 (EU numbering) in the Fc region. Cysteine ​​engineered G-CSF dimers can be produced as described, for example, in U.S. Patent No. 7,521,541. Engineered G-CSF dimer variants can provide a variety of intermolecular and intramolecular linkages.

[0231] In some embodiments, the C-terminal lysine (K447) of the Fc region can be cleaved by endogenous carboxypeptidase digestion in mammalian cell culture. The deletion 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, wherein the C-terminal lysine residue is 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, wherein the C-terminal lysine residue is cleaved.

[0232] VI. Treatment Methods

[0233] Also provided herein is a method for treating or preventing a disease or condition in an individual (e.g., a human) in need thereof or for regulating an immune response. The method comprises administering to an individual an effective amount of any G-CSF dimer pharmaceutical composition described herein (e.g., a G-CSF-Fc dimer pharmaceutical composition, such as an abegrastim α pharmaceutical composition). In some embodiments, the disease or condition is selected from neutropenia, stroke, spinal cord injury, a nervous system disease 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 to peripheral blood in allogeneic hematopoietic stem cell transplantation. In some embodiments, the disease or condition is characterized by impaired granulocyte production. In some embodiments, the disease or condition is neutropenia, such as chemotherapy-induced neutropenia or radiotherapy-induced neutropenia. In some embodiments, the disease or condition is mobilization of hematopoietic stem cells to peripheral blood in allogeneic hematopoietic 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.

[0234] In some embodiments, a method of treating or preventing a disease or disorder (e.g., a disorder characterized by impaired granulocyte production) in an individual (e.g., a human, such as a human suffering from cancer) is provided, the method comprising administering to the individual an effective amount of a pharmaceutical composition, wherein the pharmaceutical composition comprises (or consists essentially of, or consists of): (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) of a G-CSF dimer (e.g., a G-CSF-Fc dimer, such as abegrastim alfa); (b) about 1 mM to about 50 mM (e.g., about 1 mM to about 30 mM, or about 10 mM) of a buffer (e.g., sodium acetate); (c) about 0.1 mM to about 10 mM of a 5-hydroxy-1-piperidinol disulfide; and (d) about 0.1 mM to about 0.2 mM of a 5-hydroxy-1-piperidinol disulfide. (e.g., about 20 mM (e.g., 0.1 mM to about 10 mM, or about 1 mM) of a stabilizer (e.g., EDTA); (d) about 1% (w / v) to about 15% (e.g., about 1% (w / v) to about 10% (w / v), such as about 5% (w / v) or about 9% (w / v)) of a tonicity 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); wherein 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 for treating or preventing a disease or disorder (e.g., a disorder characterized by impaired leukocyte production) in an individual (e.g., a human, such as a human with cancer) is provided, the method comprising administering to the individual an effective amount of a pharmaceutical composition, 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, such as abegrastim 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; 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 of treating or preventing a disease or disorder (e.g., a disorder characterized by impaired leukocyte production) in an individual (e.g., a human, such as a human with cancer) is provided, comprising administering to the individual an effective amount of a pharmaceutical composition, wherein the pharmaceutical composition comprises (or consists essentially of or consists of): (a) about 20 mg / mL of a G-CSF dimer (e.g., a G-CSF-Fc dimer, such as abegrastim α); (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; wherein the pH of the pharmaceutical composition is about 5.2. In some embodiments, the G-CSF dimer comprises two monomer subunits, each monomer subunit comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the G-CSF dimer comprises two monomer subunits, wherein each monomer subunit comprises a G-CSF monomer (e.g., SEQ ID NO: 1), an Fc fragment (e.g., any one of SEQ ID NOs: 2-5), and an optional linker connecting the G-CSF monomer and the Fc fragment (e.g., SEQ ID: 12). In some embodiments, the G-CSF dimer comprises two monomer subunits, each subunit comprising (or consisting essentially of or consisting of): an amino acid sequence of any one of SEQ ID NOs: 6-10 (e.g., SEQ ID NO: 6). In some embodiments, the G-CSF dimer is abegrastim alpha. In some embodiments, the pharmaceutical composition is contained in a syringe (e.g., sterile and / or disposable). In some embodiments, the volume of the pharmaceutical composition within 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-induced neutropenia. In some embodiments, the method comprises administering an effective amount of a pharmaceutical composition to an individual after (e.g., 24 hours or 48 hours after) the chemotherapeutic agent is administered 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 chemotherapeutic agent is administered to the individual for at least 4 cycles (e.g., about 21 days per cycle), and wherein in each of the at least 4 cycles, the pharmaceutical composition is administered after (e.g., 24 hours or 48 hours after) the chemotherapeutic agent is administered. 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).

[0235] The methods described herein can be used to treat and / or prevent one or more conditions caused by or associated with chemotherapy, including, but not limited to, 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 greater), a decrease in the number of granulocytes, a decrease in stem cell production, a decrease in hematopoiesis, a decrease in the number of hematopoietic progenitor cells (HPCs), and conditions characterized by impaired leukocyte production.

[0236] In some embodiments, the disease or condition is characterized by impaired leukocyte production. In some embodiments, the condition characterized by impaired leukocyte production is neutropenia. In some embodiments, the condition characterized by impaired leukocyte production is chemotherapy-induced neutropenia or radiation-induced neutropenia. In some embodiments, the neutropenia is drug-induced neutropenia. In some embodiments, the neutropenia is anticancer drug-induced neutropenia. In some embodiments, the anticancer drug is myelosuppressive. In some embodiments, the neutropenia is induced by a myelosuppressive anticancer drug.

[0237] In some embodiments, a method of treating or preventing a condition characterized by impaired leukocyte production (e.g., chemotherapy-induced neutropenia or infection) in an individual (e.g., a human, such as a human with cancer) is provided, comprising administering to the individual an effective amount of any pharmaceutical composition described herein (e.g., a G-CSF-Fc dimer pharmaceutical composition, such as an abegrastim alfa pharmaceutical composition) after (e.g., at least 24 hours after) administering a chemotherapeutic agent to the individual. In some embodiments, a method of treating or preventing a condition characterized by impaired leukocyte production (e.g., chemotherapy-induced neutropenia or infection) in an individual (e.g., a human, such as a human with cancer) is provided, comprising administering to the individual an effective amount of a pharmaceutical composition after (e.g., at least 24 hours after) administration of a chemotherapeutic agent to the individual, wherein the pharmaceutical composition comprises (or consists essentially of or consists of): (a) about 20 mg / mL of a G-CSF dimer (e.g., a G-CSF-Fc dimer, such as abegrastim 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; wherein the pH of the pharmaceutical composition is about 5.2. In some embodiments, a method of 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 the following in an individual in need thereof (e.g., a human, such as an individual with cancer): i) ANC, ii) the number of granulocytes (e.g., in an individual eligible for a bone marrow transplant), iii) stem cell production, iv) hematopoiesis, and v) the number of HPCs is provided, the method comprising administering to the individual an effective amount of any pharmaceutical composition described herein (e.g., a G-CSF-Fc dimer pharmaceutical composition, such as an abegrastim alfa pharmaceutical composition) after (e.g., at least 24 hours after) administering a chemotherapeutic agent to the individual. In some embodiments, a method of reducing (e.g., reducing by at least about any of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or greater) the duration of chemotherapy-induced neutropenia (e.g., Grade 4) in an individual (e.g., a human, such as an individual with cancer) in need thereof is provided, the method comprising administering to the individual an effective amount of any pharmaceutical composition described herein (e.g., a G-CSF-Fc dimer pharmaceutical composition, such as an abegrastim alfa pharmaceutical composition) after (e.g., at least 24 hours after) administering a chemotherapeutic agent to the individual.In some embodiments, a method of reducing (e.g., reducing by at least about any of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more) or preventing the incidence of chemotherapy-induced neutropenia (e.g., Grade 4) in an individual in need thereof (e.g., a human, such as an individual with cancer) is provided, the method comprising administering to the individual an effective amount of any pharmaceutical composition described herein (e.g., a G-CSF-Fc dimer pharmaceutical composition, such as an abegrastim alfa pharmaceutical composition) after administration of a chemotherapeutic agent to the individual (e.g., at least 24 hours later). In some embodiments, the method reduces the incidence of chemotherapy-induced neutropenia (e.g., Grade 4) in an individual to less than about 0.5×10 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, 5.5, 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 prevents the ANC in the individual from reaching less than about 0.5×10 9 / L (e.g., less than about 0.4×10 9 / L, 0.2×10 9 / L, 0.1×10 9 / L, 1.0×10 7 In some embodiments, administration of the G-CSF dimer pharmaceutical composition reduces ANC from less than about 0.5×10 / L at first appearance to less than about 0.5×10 / L at first appearance 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 increases to greater than or equal to about 2.0×10 9 In some embodiments, administration of G-CSF dimer reduces ANC from less than about 0.5×10-1 to less than about 0.5×10-1 within about 10 days (e.g., within about any of 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.2, 0.1 days, or less). 9 / L increases to greater than or equal to about 2.0×10 9 / L. In some embodiments, a method of reducing (e.g., reducing by at least about any of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or greater) or preventing the incidence of febrile neutropenia (FN) in an individual in need thereof (e.g., a human, such as an individual with cancer) is provided, the method comprising administering to the individual an effective amount of any pharmaceutical composition described herein (e.g., a G-CSF-Fc dimer pharmaceutical composition, such as an abegrastim alfa pharmaceutical composition) after (e.g., at least 24 hours after) administering a chemotherapeutic agent to the individual. In some embodiments, a method of activating one or more of the following in an individual in need thereof (e.g., a human, such as an individual with cancer) is provided, the method comprising administering to the individual an effective amount of any pharmaceutical composition described herein (e.g., a G-CSF-Fc dimer pharmaceutical composition, such as abegrastim alpha pharmaceutical composition) after administration of a chemotherapeutic agent to the individual (e.g., at least 24 hours later). In some embodiments, the pharmaceutical composition comprises (or consists essentially of, or consists of): (a) about 20 mg / mL of a G-CSF dimer (e.g., a G-CSF-Fc dimer, such as abegrastim alpha); (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; wherein the pH of the pharmaceutical composition is about 5.2. In some embodiments, the G-CSF dimer comprises two monomer subunits, each subunit comprising (or consisting essentially of, or consisting of): the amino acid sequence of any one of SEQ ID NOs: 6-10 (e.g., SEQ ID NO: 6). In some embodiments, the G-CSF dimer is abegrastim alpha. In some embodiments, the method comprises administering to the individual an effective amount of the pharmaceutical composition at least 24 hours after administering the 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 epirubicin, docetaxel, cyclophosphamide, doxorubicin, etoposide, cisplatin, paclitaxel, topotecan, vincristine, methylprednisolone, cytarabine, and combinations thereof. In some embodiments, two or more chemotherapy drugs are administered to the individual, 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 to treat cancer.In some embodiments, the cancer is selected from 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 is administered at least 4 cycles of chemotherapeutic agents, and wherein the pharmaceutical composition is administered after the chemotherapeutic agent is administered in each cycle of at least 4 cycles (e.g., at least 24 hours). In some embodiments, the individual suffers from breast cancer, (i) wherein the individual is administered at least 4 cycles of docetaxel and cyclophosphamide, wherein docetaxel 75 mg / m is administered on day 1 of each cycle. 2 and cyclophosphamide 600 mg / m 2 , and wherein the pharmaceutical composition is administered on day 2 of each cycle (e.g., at least 24 hours after administration of docetaxel and cyclophosphamide); (ii) wherein the individual is administered at least 4 cycles of epirubicin and cyclophosphamide, wherein epirubicin 100 mg / m 2 and cyclophosphamide 600 mg / m 2 , and wherein 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) wherein the subject is administered at least 4 cycles of docetaxel and doxorubicin, wherein docetaxel 75 mg / m is administered on day 1 of each cycle 2 and doxorubicin 60 mg / m 2 , and wherein the pharmaceutical composition is administered on the 2nd day 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, for each administration, the pharmaceutical composition is administered with about 5 mg to about 25 mg (e.g., about 10 mg / about 25 mg, such as about 20 mg) of G-CSF dimer. In some embodiments, the pharmaceutical composition is administered subcutaneously. In some embodiments, the pharmaceutical composition is contained (e.g., pre-filled) in a syringe (e.g., sterile and / or disposable). In some embodiments, the volume of the pharmaceutical composition in the syringe is about 1 mL.

[0238] In some embodiments, a method of treating cancer (e.g., breast cancer) in an individual (e.g., a human) is provided, the method comprising: administering an effective amount of a chemotherapeutic agent to the individual, and further administering to the individual any pharmaceutical composition described herein (e.g., a G-CSF-Fc dimer pharmaceutical composition, such as an abegrastim alpha pharmaceutical composition) after the administration of the chemotherapeutic agent to the individual (e.g., at least 24 hours later), wherein the administration of the pharmaceutical composition alleviates or prevents a condition characterized by impaired leukocyte production (e.g., chemotherapy-induced neutropenia or infection). In some embodiments, the condition characterized by impaired leukocyte 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 epirubicin, docetaxel, cyclophosphamide, doxorubicin, etoposide, cisplatin, paclitaxel, topotecan, vincristine, methylprednisolone, cytarabine, and combinations thereof. In some embodiments, two or more chemotherapy drugs are administered to an individual, 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, two or more chemotherapy drugs are administered simultaneously (e.g., in the same formulation or in separate formulations). In some embodiments, two or more chemotherapy drugs are administered sequentially. In some embodiments, cancer is selected from 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 a G-CSF dimer pharmaceutical composition increases (e.g., increases by at least about any of 10%, 20%, 50%, 70%, 90%, 1-fold, 2-fold, 5-fold, 20-fold, or greater) one or more of i) ANC, ii) granulocyte number (e.g., in a subject eligible for a bone marrow transplant), iii) stem cell production, iv) hematopoiesis, and v) the number of HPCs in an individual. In some embodiments, administration of a G-CSF dimer pharmaceutical composition decreases (e.g., decreases by at least about any of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or greater) 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., reduces by at least about any of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or greater) or prevents the incidence of chemotherapy-induced neutropenia (e.g., Grade 4) in a subject. In some embodiments, administration of the G-CSF dimer pharmaceutical composition reduces the incidence of chemotherapy-induced neutropenia (e.g., Grade 4) in a subject by less than about 0.5×10 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, 5.5, 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, administration of the G-CSF dimer pharmaceutical composition prevents the ANC in a subject from reaching less than about 0.5×10 9 / L (for example, less than 0.4×10 9 / L, 0.2×10 9 / L, 0.1×10 9 / 1L, 1.0×10 7 In some embodiments, administration of the G-CSF dimer pharmaceutical composition reduces ANC from less than about 0.5×10-1 to less than about 0.5×10-1 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 increases to greater than or equal to about 2.0×10 9 / L. In some embodiments, administration of a G-CSF dimer pharmaceutical composition reduces (e.g., reduces by at least about any of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or greater) or prevents the incidence of FN in an individual. In some embodiments, administration of a G-CSF dimer pharmaceutical composition activates one or more of: i) neutrophil precursor cells, ii) bone marrow stem cells, and iii) mature neutrophils in an individual. In some embodiments, the method of treating cancer can achieve one or more of the following biological activities: (1) killing (e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) of cancer cells; (2) inhibiting (e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) the proliferation of cancer cells; (3) reducing (e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) of tumor size; (4) reduce (e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) one or more symptoms in a subject with cancer; (5) inhibit (e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) the progression of cancer; (6) reduce (e.g., by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) the manifestation, incidence, or burden of pre-existing tumor metastases (e.g., metastases to lymph nodes); (7) prolong survival, e.g., prolong the survival of a subject by at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 100%. (8) prolonging the time to cancer progression, for example, by at least about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 60 weeks or longer; and (9) preventing, inhibiting, or reducing (for example, 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 mg / mL of a G-CSF dimer (e.g., a G-CSF-Fc dimer such as abegrastim alpha); (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; wherein the pH of the pharmaceutical composition is about 5.2. In some embodiments, the G-CSF dimer comprises two monomer subunits, each monomer subunit comprising (or consisting essentially of, or consisting of): the amino acid sequence of any one of SEQ ID NOs: 6-10 (e.g., SEQ ID NO: 6). In some embodiments, the G-CSF dimer is abegrastim alpha. In some embodiments, the pharmaceutical composition is administered at about 5 mg to about 25 mg (e.g., about 10 mg / about 25 mg, such as about 20 mg) of G-CSF dimer for each administration. In some embodiments, the pharmaceutical composition is administered subcutaneously. In some embodiments, the pharmaceutical composition is contained (e.g., prefilled) in a syringe (e.g., sterile and / or disposable). In some embodiments, the volume of the pharmaceutical composition in the syringe is about 1 mL.

[0239] The G-CSF dimer (e.g., G-CSF-Fc dimer, such as abegrastim alfa) pharmaceutical composition can be administered to the individual after the chemotherapeutic agent is administered, for example, at least 24 hours after the chemotherapeutic agent is administered. In some embodiments, the pharmaceutical composition is administered 24, 28, 30, 32, 36, 40, 44, 48, 52, 60, 72 or more after the chemotherapeutic agent is administered, for example, 24 hours or 48 hours after the chemotherapeutic agent is administered.

[0240] In some embodiments, when an individual is administered 2 or more (e.g., 4) cycles of a chemotherapeutic agent, the G-CSF dimer pharmaceutical composition is administered after the last dose of the chemotherapeutic agent in the cycle (e.g., at least 24 hours later). For example, if the chemotherapeutic agent is administered at one dose on day 1 of the cycle, the G-CSF dimer pharmaceutical composition is administered after the one dose of the chemotherapeutic agent is administered in the cycle (e.g., at least 24 hours later). If the chemotherapeutic agent is administered at a first dose on day 1 of a cycle (i.e., a cycle of at least 2 days) and a second dose on day 2, the G-CSF dimer pharmaceutical composition is administered after the second dose of the chemotherapeutic agent is administered in the cycle (e.g., at least 24 hours later). Similarly, if an individual is administered 2 or more doses (e.g., 4 doses) of a chemotherapeutic agent in only one cycle, the G-CSF dimer pharmaceutical composition is administered after the last dose of the chemotherapeutic agent is administered (e.g., at least 24 hours later). In some embodiments, when an individual is administered 2 or more (e.g., 4) cycles of a chemotherapeutic agent, for each cycle, administration of the G-CSF dimer pharmaceutical composition after administration of the chemotherapeutic agent (e.g., at least 24 hours) is simultaneous, for example, the G-CSF dimer pharmaceutical composition is administered 24 hours or 48 hours after administration of the chemotherapeutic agent in each cycle. In some embodiments, when an individual is administered 2 or more (e.g., 4) cycles of a chemotherapeutic agent, administration of the G-CSF dimer pharmaceutical composition after administration of the chemotherapeutic agent (e.g., at least 24 hours) may be at different times in 2 or more cycles, for example, 24 hours after administration of the chemotherapeutic agent in one cycle and 48 hours after administration of the chemotherapeutic agent in another cycle. In some embodiments, when an individual is administered 2 or more (e.g., 4) cycles of a chemotherapeutic agent, administration of the G-CSF dimer pharmaceutical composition is after administration of the chemotherapeutic agent (e.g., at least 24 hours later) in at least cycle 1 (e.g., cycle 1, cycles 1-4, or all cycles) .

[0241] In some embodiments, the subject is administered two or more chemotherapeutic agents (e.g., docetaxel and cyclophosphamide), for example, two or more chemotherapeutic agents per cycle. In some embodiments, two or more chemotherapeutic agents are administered simultaneously in the same formulation or separate formulations. In some embodiments, two or more chemotherapeutic agents are administered sequentially, for example, the first chemotherapeutic agent is administered on day 1 and the second chemotherapeutic agent is administered on day 2. When the subject is administered two or more chemotherapeutic agents sequentially, in some embodiments, the administration of the G-CSF dimer pharmaceutical composition is after the last chemotherapeutic agent (e.g., the last chemotherapeutic agent of the cycle) is administered (e.g., at least 24 hours later). In some embodiments, when two or more chemotherapeutic agents are administered at least 2 days apart, for example, the first chemotherapeutic agent is administered on day 1 and the second chemotherapeutic agent is administered on day 3 or later, depending on the ANC level and / or other conditions (e.g., infection, fever, vital signs, laboratory measurements) of the individual to be treated, administration of the G-CSF dimer pharmaceutical composition can be performed after administration of the first chemotherapeutic agent (e.g., at least 24 hours later), or the G-CSF dimer pharmaceutical composition can be performed after administration of each of the two or more chemotherapeutic agents (e.g., at least 24 hours later).

[0242] In some embodiments, the subject is administered at least 2 (e.g., 2, 3, or 4) cycles of a chemotherapeutic agent. In some embodiments, the G-CSF dimer pharmaceutical composition is administered after (e.g., at least 24 hours after) the chemotherapeutic agent is administered in cycle 1. In some embodiments, the subject is administered at least 4 cycles of a chemotherapeutic agent, wherein the G-CSF dimer pharmaceutical composition is administered after (e.g., at least 24 hours after) the chemotherapeutic agent is administered in cycle 1. Thus, in some embodiments, a method for treating or preventing a condition characterized by impaired leukocyte production (e.g., chemotherapy-induced neutropenia or infection) in an individual in need thereof (e.g., a human, such as an individual with cancer) is provided, wherein the individual is administered at least 2 (e.g., 2, 3, or 4) cycles of a chemotherapeutic agent (e.g., the chemotherapeutic agent is administered on day 1 of each cycle), wherein the method comprises administering to the individual an effective amount of any pharmaceutical composition described herein (e.g., a G-CSF-Fc dimer pharmaceutical composition, such as an abegrastim alfa pharmaceutical composition) after (e.g., at least 24 hours after) the chemotherapeutic agent is administered in cycle 1. In some embodiments, a method for treating or preventing a condition characterized by impaired leukocyte production (e.g., chemotherapy-induced neutropenia or infection) in an individual in need thereof (e.g., a human, such as an individual with cancer) is provided, wherein the individual is administered at least 4 cycles (e.g., 4 cycles) of a chemotherapeutic agent (e.g., the chemotherapeutic agent is administered on day 1 of each cycle), wherein the method comprises administering to the individual an effective amount of any pharmaceutical composition described herein (e.g., a G-CSF-Fc dimer pharmaceutical composition, such as an abegrastim alfa pharmaceutical composition) after (e.g., at least 24 hours after) administration of the chemotherapeutic agent in cycle 1. In some embodiments, the individual is administered a chemotherapeutic agent to treat cancer (e.g., breast cancer). In some embodiments, a method for treating cancer (e.g., breast cancer) in an individual (e.g., a human) is provided, the method comprising: i) administering an effective amount of a chemotherapeutic agent to the individual for at least 2 (e.g., 2, 3, or 4) cycles (e.g., administering the chemotherapeutic agent on day 1 of each cycle); and ii) administering an effective amount of any pharmaceutical composition described herein (e.g., a G-CSF-Fc dimer pharmaceutical composition, such as an abegrastim alfa pharmaceutical composition) to the individual after administration of the chemotherapeutic agent in cycle 1 (e.g., at least 24 hours later); wherein administration of the pharmaceutical composition alleviates or prevents a condition characterized by impaired leukocyte production (e.g., chemotherapy-induced neutropenia or infection) in at least one of the 2 or more cycles (e.g., cycle 1 or cycles 1-2).In some embodiments, a method for treating cancer (e.g., breast cancer) in an individual (e.g., a human) is provided, the method comprising: i) administering an effective amount of a chemotherapeutic agent to the individual for at least 4 cycles (e.g., 4 cycles) (e.g., administering the chemotherapeutic agent on day 1 of each cycle); and ii) administering an effective amount of any pharmaceutical composition described herein (e.g., a G-CSF-Fc dimer pharmaceutical composition, such as an abegrastim alfa pharmaceutical composition) to the individual after the administration of the chemotherapeutic agent in cycle 1 (e.g., at least 24 hours later); wherein administration of the pharmaceutical composition alleviates or prevents a condition characterized by impaired leukocyte production (e.g., chemotherapy-induced neutropenia or infection) in at least one of the 4 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 later or 48 hours later) after the 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 after the administration of the chemotherapeutic agent in each cycle starting cycle 2 (e.g., in each cycle of at least cycles 2-4) (e.g., 24 hours or any one of 26, 28, 30, 32, 36, 40, 44, 48, 60 hours or more). In some embodiments, the G-CSF dimer pharmaceutical composition is administered 48 hours after the administration of the chemotherapeutic agent in each cycle starting cycle 2 (e.g., in each cycle of at least cycles 2-4) (e.g., 48 hours or 48 hours). In some embodiments, the chemotherapeutic agent is administered on day 1 of each cycle (e.g., 2 days or more of each cycle). In some embodiments, each cycle is about 21 days. In some embodiments, the administration time of the G-CSF dimer pharmaceutical composition is the same in each cycle starting cycle 2 (e.g., in each cycle of at least cycles 2-4). In some embodiments, the administration time of the G-CSF dimer pharmaceutical composition is different in 2 or more cycles starting with cycle 2, for example, different in at least 2 or more cycles of cycles 2-4. In some embodiments, the disorder characterized by impaired leukocyte 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., administered 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 mg / mL of a G-CSF dimer (e.g., a G-CSF-Fc dimer, such as abegrastim α); (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; wherein the pH of the pharmaceutical composition is about 5.2.In some embodiments, the G-CSF dimer comprises two monomer subunits, each monomer subunit comprising (or consisting essentially of, or consisting of): the amino acid sequence of any one of SEQ ID NOs: 6-10 (e.g., SEQ ID NO: 6). In some embodiments, the G-CSF dimer is abegrastim α. In some embodiments, the pharmaceutical composition is administered at about 5 mg to about 25 mg (e.g., about 10 mg / about 25 mg, such as about 20 mg) of G-CSF dimer for each administration. In some embodiments, the pharmaceutical composition is administered subcutaneously. In some embodiments, the pharmaceutical composition is contained (e.g., pre-filled) in a syringe (e.g., sterile and / or disposable). In some embodiments, the volume of the pharmaceutical composition within the syringe is about 1 mL. In some embodiments, administration of a G-CSF dimer pharmaceutical composition achieves one or more of the following effects in one or more cycles (e.g., cycle 1, cycle 1-2, cycle 2-4, or cycle 1-4): i) shortening the duration of chemotherapy-induced neutropenia; ii) reducing or preventing the incidence of chemotherapy-induced neutropenia; iii) reducing or preventing the incidence of FN; iv) increasing ANC; v) activating neutrophil precursor cells; vi) activating bone marrow stem cells; vii) activating mature neutrophils; viii) reducing the number of FN in an individual to less than about 0.5×10. 9 / L to less than about 36 hours; ix) preventing the ANC in the individual from reaching less than about 0.5×10 9 / L; x) ANC decreased from less than about 0.5×10 9 / L increases to greater than or equal to about 2.0×10 9 / L; xi) improving the survival rate of the individual. In some embodiments, the chemotherapy-induced neutropenia is grade 4 neutropenia.

[0243] In some embodiments, the subject is administered at least 2 (e.g., 2, 3, or 4) cycles of a chemotherapeutic agent, wherein the G-CSF dimer pharmaceutical composition is administered after (e.g., at least 24 hours after) the chemotherapeutic agent is administered in each of the at least 2 cycles. In some embodiments, the subject is administered at least 4 cycles of a chemotherapeutic agent, wherein the G-CSF dimer pharmaceutical composition is administered after (e.g., at least 24 hours after) the chemotherapeutic agent is administered in each of the at least 4 cycles. Thus, in some embodiments, a method for treating or preventing a condition characterized by impaired leukocyte production (e.g., chemotherapy-induced neutropenia or infection) in an individual in need thereof (e.g., a human, such as an individual with cancer) is provided, wherein the individual is administered at least 2 (e.g., 2, 3, or 4) cycles of a chemotherapeutic agent (e.g., the chemotherapeutic agent is administered on day 1 of each cycle), and wherein the method comprises administering to the individual an effective amount of any pharmaceutical composition described herein (e.g., a G-CSF-Fc dimer pharmaceutical composition, e.g., abegrastim alfa pharmaceutical composition) after (e.g., at least 24 hours after) administration of the chemotherapeutic agent in each of the at least 2 cycles. In some embodiments, a method for treating or preventing a condition characterized by impaired leukocyte production (e.g., chemotherapy-induced neutropenia or infection) in an individual in need thereof (e.g., a human, such as an individual with cancer) is provided, wherein the individual is administered at least 4 cycles (e.g., 4 cycles) of a chemotherapeutic agent (e.g., the chemotherapeutic agent is administered on day 1 of each cycle), and wherein the method comprises administering to the individual an effective amount of any pharmaceutical composition described herein (e.g., a G-CSF-Fc dimer pharmaceutical composition, e.g., abegrastim alfa pharmaceutical composition) after administration of the chemotherapeutic agent in each of the at least 4 cycles (e.g., at least 24 hours later). In some embodiments, the individual is administered a chemotherapeutic agent to treat cancer (e.g., breast cancer). In some embodiments, a method for treating cancer (e.g., breast cancer) in an individual (e.g., a human, such as an individual suffering from cancer) is provided, the method comprising: i) administering to the individual an effective amount of a chemotherapeutic agent for at least 2 (e.g., 2, 3, or 4) cycles (e.g., administering the chemotherapeutic agent on day 1 of each cycle); and ii) administering to the individual an effective amount of any pharmaceutical composition described herein (e.g., a G-CSF-Fc dimer pharmaceutical composition, such as an abegrastim alfa pharmaceutical composition) after administration of the chemotherapeutic agent in each of the at least 2 cycles (e.g., at least 24 hours later); wherein administration of the pharmaceutical composition alleviates or prevents a condition characterized by impaired leukocyte production (e.g., chemotherapy-induced neutropenia or infection) in at least one of the 2 or more cycles (e.g., cycle 1 or cycles 1-2).In some embodiments, a method for treating cancer (e.g., breast cancer) in an individual (e.g., a human, such as an individual with cancer) is provided, the method comprising: i) administering an effective amount of a chemotherapeutic agent to the individual for at least 4 cycles (e.g., 4 cycles) (e.g., administering the chemotherapeutic agent on day 1 of each cycle); and ii) administering an effective amount of any pharmaceutical composition described herein (e.g., a G-CSF-Fc dimer pharmaceutical composition, such as an abegrastim alpha pharmaceutical composition) to the individual after the administration of the chemotherapeutic agent in each of the at least 4 cycles (e.g., at least 24 hours later); wherein administration of the pharmaceutical composition alleviates or prevents a condition characterized by impaired leukocyte production (e.g., chemotherapy-induced neutropenia or infection) in at least one of the 4 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., 2 or more days per cycle). In some embodiments, each cycle is about 21 days. In some embodiments, the administration time of the G-CSF dimer pharmaceutical composition is the same in each of the at least 2 cycles. In some embodiments, the administration time of the G-CSF dimer pharmaceutical composition is different in each of the at least 2 cycles. In some embodiments, the administration time of the G-CSF dimer pharmaceutical composition is the same in each of at least 4 cycles. In some embodiments, the administration time of the G-CSF dimer pharmaceutical composition is different in two or more cycles (e.g., 4) of at least 4 cycles. In some embodiments, in each of at least 2 cycles, for example, in each of at least 4 cycles, the G-CSF dimer pharmaceutical composition is administered at least 24 hours (e.g., 24 hours later or 48 hours later) after the administration of the chemotherapeutic agent. In some embodiments, the disorder characterized by impaired leukocyte production is chemotherapy-induced neutropenia. In some embodiments, the chemotherapeutic agent is a bone marrow suppressive chemotherapeutic agent. In some embodiments, two chemotherapeutic drugs are administered to an individual in each cycle, for example, simultaneously (e.g., administered in the same formulation or a separate formulation). In some embodiments, the pharmaceutical composition comprises (or consists essentially of, or consists of): (a) about 20 mg / mL of a G-CSF dimer (e.g., a G-CSF-Fc dimer, such as abegrastim α); (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; wherein the pH of the pharmaceutical composition is about 5.2. In some embodiments, the G-CSF dimer comprises two monomer subunits, each of which comprises (or consists essentially of, or consists of): the amino acid sequence of any one of SEQ ID NOs: 6-10 (e.g., SEQ ID NO: 6).In some embodiments, the G-CSF dimer is abegrastim alpha. In some embodiments, the pharmaceutical composition is administered at about 5 mg to about 25 mg (e.g., about 10 mg / about 25 mg, such as about 20 mg) of G-CSF dimer for each administration. In some embodiments, the pharmaceutical composition is administered subcutaneously. In some embodiments, the pharmaceutical composition is contained (e.g., prefilled) in a syringe (e.g., sterile and / or disposable). In some embodiments, the volume of the pharmaceutical composition in the syringe is about 1 mL. In some embodiments, administration of a G-CSF dimer pharmaceutical composition achieves one or more of the following effects in one or more cycles (e.g., cycle 1, cycle 1-2, cycle 2-4, or cycle 1-4): i) shortening the duration of chemotherapy-induced neutropenia; ii) reducing or preventing the incidence of chemotherapy-induced neutropenia; iii) reducing or preventing the incidence of FN; iv) increasing ANC; v) activating neutrophil precursor cells; vi) activating bone marrow stem cells; vii) activating mature neutrophils; viii) reducing the number of FN in an individual to less than about 0.5×10. 9 / L to less than about 36 hours; ix) preventing the ANC in the individual from reaching less than about 0.5×10 9 / L; x) ANC decreased from less than about 0.5×10 9 / L increases to greater than or equal to about 2.0×10 9 / L; xi) improving the survival rate of the individual. In some embodiments, the chemotherapy-induced neutropenia is grade 4 neutropenia.

[0244] In some embodiments, a method for treating or preventing a condition characterized by impaired leukocyte production (e.g., chemotherapy-induced neutropenia or infection) in an individual (e.g., a human) in need thereof is provided, wherein the method comprises administering an effective amount of any pharmaceutical composition described herein (e.g., a G-CSF-Fc dimer pharmaceutical composition, such as abegrastim alfa) after administering one or more chemotherapeutic agents in each of at least 4 cycles, wherein the individual has breast cancer (e.g., metastatic or non-metastatic), and (i) wherein the individual is administered at least 4 cycles of docetaxel and cyclophosphamide, wherein docetaxel 75 mg / m is administered on day 1 of each cycle. 2 and cyclophosphamide 600 mg / m 2 , and wherein the pharmaceutical composition is administered on day 2 of each cycle at least 24 hours after administration of docetaxel and cyclophosphamide; (ii) wherein the individual is administered at least 4 cycles of epirubicin and cyclophosphamide, wherein epirubicin 100 mg / m 2and cyclophosphamide 600 mg / m 2 , and wherein the pharmaceutical composition is administered on day 3 of each cycle at least 48 hours after administration of epirubicin and cyclophosphamide; or (iii) wherein the subject is administered at least 4 cycles of docetaxel and doxorubicin, wherein docetaxel 75 mg / m is administered on day 1 of each cycle 2 and doxorubicin 60 mg / m 2 , and wherein the pharmaceutical composition is administered on day 2 of each cycle at least 24 hours after 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 is provided, wherein the method comprises administering one or more chemotherapeutic agents in each cycle of at least 4 cycles, and further administering an effective amount of any pharmaceutical composition described herein (e.g., a G-CSF-Fc dimer pharmaceutical composition, such as an abegrastim α pharmaceutical composition) after administering the one or more chemotherapeutic agents in each cycle of the at least 4 cycles, wherein administration of the pharmaceutical composition alleviates or prevents a condition characterized by impaired leukocyte production (e.g., chemotherapy-induced neutropenia or infection); and (i) wherein the individual is administered at least 4 cycles of docetaxel and cyclophosphamide, wherein docetaxel 75 mg / m is administered on day 1 of each cycle 2 and cyclophosphamide 600 mg / m 2 , and wherein the pharmaceutical composition is administered on day 2 of each cycle at least 24 hours after administration of docetaxel and cyclophosphamide; (ii) wherein the subject is administered at least 4 cycles of epirubicin and cyclophosphamide, wherein epirubicin 100 mg / m 2 and cyclophosphamide 600 mg / m 2 , and wherein the pharmaceutical composition is administered on day 3 of each cycle at least 48 hours after administration of epirubicin and cyclophosphamide; or (iii) wherein the subject is administered at least 4 cycles of docetaxel and doxorubicin, wherein docetaxel 75 mg / m 2 and doxorubicin 60 mg / m 2, and wherein the pharmaceutical composition is administered on day 2 of each cycle 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 comprises (or consists essentially of, or consists of): (a) about 20 mg / mL of a G-CSF dimer (e.g., a G-CSF-Fc dimer, such as abegrastim α); (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; wherein the pH of the pharmaceutical composition is about 5.2. In some embodiments, the G-CSF dimer comprises two monomer subunits, each monomer subunit comprising (or consisting essentially of, or consisting of): the amino acid sequence of any one of SEQ ID NOs: 6-10 (e.g., SEQ ID NO: 6). In some embodiments, the G-CSF dimer is abegrastim alpha. In some embodiments, the pharmaceutical composition is administered at about 5 mg to about 25 mg (e.g., about 10 mg / about 25 mg, such as about 20 mg) of G-CSF dimer for each administration. In some embodiments, the pharmaceutical composition is administered subcutaneously. In some embodiments, the pharmaceutical composition is contained (e.g., prefilled) in a syringe (e.g., sterile and / or disposable). In some embodiments, the volume of the pharmaceutical composition in the syringe is about 1 mL.

[0245] 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 such as an abegrastim alfa pharmaceutical composition) after administration of a chemotherapeutic agent to a subject (e.g., at least 24 hours later), induces an increase (e.g., an increase of at least about any of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 1-fold, 2-fold, 5-fold, 10-fold, 50-fold, or greater) in white blood cells (WBC) or a decrease (e.g., a decrease of about any of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or greater) in WBC loss in a subject. For example, in some embodiments, the number of neutrophils (e.g., ANC) in an individual is increased (e.g., increased 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 the number of neutrophils in an individual is inhibited (e.g., inhibited by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more). In some embodiments, the minimum ANC of an individual is increased (e.g., increased 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 ANC in the individual is increased (e.g., increased 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 greater). In some embodiments, the time of ANC recovery in the individual is reduced (e.g., reduced by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or greater). In some embodiments, one or more effects described herein are achieved in cycle 1 of chemotherapy for the individual. In some embodiments, one or more effects described herein are achieved in one or more cycles of chemotherapy for the individual (e.g., cycles 1-2 of 2 or more cycles, or cycles 1-3 of 4 or more cycles). In some embodiments, one or more effects described herein are achieved in each cycle of chemotherapy for the individual (e.g., all 2 cycles or all 4 cycles).

[0246] 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, such as an abegrastim alfa pharmaceutical composition) after administration of a chemotherapeutic agent to an individual (e.g., at least 24 hours later) reduces (e.g., by at least about any of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or greater) the duration of chemotherapy-induced neutropenia (e.g., Grade 4) in an individual. In some embodiments, for one or more grades of neutropenia (e.g., Grade I, II, III, or IV), the duration of chemotherapy-induced neutropenia is shortened. In some embodiments, the methods described herein reduce the duration of Grade 4 (severe) neutropenia, or reduce it by less than about 0.5×10 9 / L of ANC, for example, to less than about 36 hours, for example, to 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, the methods described herein shorten the duration of Grade 4 (severe) neutropenia, or to less than about 0.5×10 9 / L is shortened to less than about 36 hours. In some embodiments, Grade 4 (severe) neutropenia (ANC <0.5×10 9 The duration of Grade 4 neutropenia in cycle 1 of chemotherapy for an individual is shortened by less than about 0.5×109 / L. 9 In some embodiments, the methods described herein shorten the duration of Grade 4 neutropenia in one or more cycles of chemotherapy (e.g., cycles 1-2 in 2 or more cycles, or cycles 1-3 in 4 or more cycles) of an individual, or shorten the duration of Grade 4 neutropenia by less than about 0.5×10 9 In some embodiments, the methods described herein shorten the duration of Grade 4 neutropenia in each cycle of chemotherapy (e.g., all 2 cycles or all 4 cycles) of an individual, or shorten it by less than about 0.5×10 9 In some embodiments, the methods described herein reduce the duration of Grade 3 neutropenia, or reduce it by less than about 1.0×10 9 / L, for example, to less than about 36 hours, for example, to 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 9 The duration of time that an individual has an ANC < 1.0 × 10 9 In some embodiments, the methods described herein shorten the duration of Grade 3 neutropenia in cycle 1 of chemotherapy in an individual, or shorten it by less than about 1.0×10 9 In some embodiments, the methods described herein shorten the duration of Grade 3 neutropenia in one or more cycles of chemotherapy (e.g., cycles 1-2 in 2 or more cycles, or cycles 1-3 in 4 or more cycles) of an individual, or shorten the duration of Grade 3 neutropenia by less than about 1.0×10 9 In some embodiments, the methods described herein shorten the duration of Grade 3 neutropenia in each cycle (e.g., all 2 cycles or all 4 cycles) of chemotherapy in an individual, or shorten it by less than about 1.0×10 9 / L duration of ANC.

[0247] 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, such as an abegrastim alfa pharmaceutical composition) after administration of a chemotherapeutic agent to an individual (e.g., at least 24 hours later) reduces (e.g., reduces by at least about any of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or greater) or prevents the incidence of chemotherapy-induced neutropenia (e.g., Grade 4) in an individual. In some embodiments, the incidence of chemotherapy-induced neutropenia is reduced for one or more grades of neutropenia, such as Grade I, Grade II, Grade III, or Grade IV. In some embodiments, the incidence of chemotherapy-induced neutropenia can be reduced or prevented for any grade of neutropenia. In some embodiments, the methods described herein reduce or prevent the incidence of Grade 4 (severe) neutropenia. In some embodiments, the methods described herein prevent an ANC in an individual from reaching less than about 0.5×10 9 / L. In some embodiments, the methods described herein reduce the incidence of Grade 4 neutropenia at least once (e.g., at least 2, 3, 4, or more times), or reduce the ANC to less than about 0.5×10 9 In some embodiments, the methods described herein reduce or prevent the incidence of Grade 4 neutropenia during cycle 1 of chemotherapy in an individual, or reduce or prevent the ANC from reaching less than about 0.5×10 9 In some embodiments, the methods described herein reduce or prevent the incidence of Grade 4 neutropenia, or reduce or prevent the ANC from reaching less than about 0.5×10 / L in one or more cycles of chemotherapy in an individual, such as 2 or more cycles of cycles 1-2, or 4 or more cycles of cycles 1-3. 9 In some embodiments, the methods described herein reduce or prevent the incidence of Grade 4 neutropenia, or reduce or prevent ANC from reaching less than about 0.5×10 / L in each cycle of chemotherapy (e.g., all 2 cycles or all 4 cycles) of the individual. 9 In some embodiments, the methods described herein reduce or prevent the incidence of Grade 3 neutropenia. In some embodiments, the methods described herein prevent the ANC in an individual from reaching less than about 1.0×10 9 / L. In some embodiments, the methods described herein reduce the incidence of Grade 3 neutropenia at least once (e.g., at least 2, 3, 4, or more times), or reduce the ANC to less than about 1.0×10 9 In some embodiments, the methods described herein reduce or prevent the incidence of Grade 3 neutropenia, or reduce or prevent ANC below about 1.0×10 9 In some embodiments, the methods described herein reduce or prevent the incidence of Grade 3 neutropenia, or reduce or prevent the ANC from reaching less than about 1.0×10 / L in one or more cycles of chemotherapy in an individual, such as 2 or more cycles 1-2, or 4 or more cycles 1-3. 9 In some embodiments, the methods described herein reduce or prevent the incidence of Grade 3 neutropenia, or reduce or prevent ANC from reaching less than about 1.0×10 / L in each cycle of chemotherapy (e.g., all 2 cycles or all 4 cycles) of an individual. 9 / L incidence.

[0248] 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, such as an abegrastim alfa pharmaceutical composition) after administration of a chemotherapeutic agent to a subject (e.g., at least 24 hours later), reduces (e.g., by at least about any of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more) or prevents the incidence of febrile neutropenia (FN) in a subject. FN is defined as a single oral temperature of ≥38.3°C (101°F) or >38.0°C (100.4°F) for >1 hour on the same day and an ANC <0.5×10 9 / L. Thus, in some embodiments, the methods described herein reduce or prevent a single oral temperature of ≥38.3°C (101°F) or >38.0°C (100.4°F) for more than 1 hour and ANC <0.5×10 9 / L. In some embodiments, the methods described herein reduce the incidence of FN at least once, for example at least 2, 3, 4 times or more. In some embodiments, the methods described herein reduce or prevent the incidence of FN in cycle 1 of chemotherapy for an individual. In some embodiments, the methods described herein reduce or prevent the incidence of FN in one or more cycles of chemotherapy for an individual (e.g., cycles 1-2 of 2 or more cycles, or cycles 1-3 of 4 or more cycles). In some embodiments, the methods described herein reduce or prevent the incidence of FN in each cycle of chemotherapy for an individual (e.g., all 2 cycles or all 4 cycles).

[0249] 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, such as an abegrastim alfa pharmaceutical composition) after administration of a chemotherapeutic agent to an individual (e.g., at least 24 hours later), reduces (e.g., reduces by at least about any of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more) the duration and / or incidence of infection (e.g., infection manifested by neutropenia, such as FN) in an individual, or prevents its occurrence. In some embodiments, the methods described herein reduce (e.g., reduces by at least about any of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more) the duration and / or incidence of use of intravenous antibiotics. In some embodiments, the methods described herein reduce the incidence of infection by at least one time, such as at least 2, 3, 4 times or more. In some embodiments, the methods described herein reduce the duration and / or incidence of infection in cycle 1 of chemotherapy for an individual, or prevent the occurrence of infection. In some embodiments, the methods described herein reduce the duration and / or incidence of infection in one or more cycles of chemotherapy for an individual (e.g., cycles 1-2 of 2 or more cycles, or cycles 1-3 of 4 or more cycles), or prevent the occurrence of infection. In some embodiments, the methods described herein reduce the duration and / or incidence of infection in each cycle of chemotherapy for an individual (e.g., all 2 cycles or all 4 cycles), or prevent the occurrence of infection.

[0250] 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, such as an abegrastim alfa pharmaceutical composition) after administration of a chemotherapeutic agent to an individual (e.g., at least 24 hours later), reduces (e.g., reduces by at least about any of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or greater) the duration and / or incidence of hospitalization for febrile neutropenia or infection in an individual, or prevents its occurrence. In some embodiments, the methods described herein reduce (e.g., reduces by at least about any of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or greater) the duration and / or incidence of use of intravenous antibiotics. In some embodiments, the methods described herein reduce the incidence of hospitalization for FN or infection by at least one time, for example, by at least 2, 3, 4 times or more. In some embodiments, the methods described herein reduce the duration and / or incidence of hospitalization for FN or infection in cycle 1 of chemotherapy for an individual, or prevent its occurrence. In some embodiments, the methods described herein reduce the duration and / or incidence of hospitalization for FN or infection in one or more cycles of chemotherapy for an individual (e.g., cycles 1-2 for 2 or more cycles, or cycles 1-3 for 4 or more cycles), or prevent its occurrence. In some embodiments, the methods described herein reduce the duration and / or incidence of hospitalization for FN or infection in each cycle of chemotherapy for an individual (e.g., all 2 cycles or all 4 cycles), or prevent its occurrence.

[0251] 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, such as an abegrastim alfa pharmaceutical composition) after administration of a chemotherapeutic agent to a subject (e.g., at least 24 hours later), reduces (e.g., by at least about any of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more) the depth of the ANC nadir in a subject, or increases (e.g., by at least about any of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 1-fold, 2-fold, 5-fold, 10-fold, or more) the ANC nadir value in a subject. In some embodiments, the methods described herein reduce the depth of the ANC nadir, or increase the ANC nadir value, in cycle 1 of chemotherapy in a subject. In some embodiments, the methods described herein reduce the depth of the ANC nadir, or increase the ANC nadir value, in one or more cycles of chemotherapy in an individual (e.g., cycles 1-2 of 2 or more cycles, or cycles 1-3 of 4 or more cycles). In some embodiments, the methods described herein reduce the depth of the ANC nadir, or increase the ANC nadir value, in each cycle of chemotherapy in an individual (e.g., all 2 cycles or all 4 cycles).

[0252] 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, such as an abegrastim alfa pharmaceutical composition) after administration of a chemotherapeutic agent to an individual (e.g., at least 24 hours later), reduces (e.g., by at least about any of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more) the time to ANC recovery in an individual. In some embodiments, the time to ANC recovery is reduced by at least about any of 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 is achieved after an ANC nadir (e.g., an expected ANC nadir) of ANC ≥ 2.0 × 10 9 / L. Thus, in some embodiments, the methods described herein shorten the ANC nadir (e.g., the first occurrence of ANC less than about 0.5×10 9 / L) to reach ANC ≥ 2.0 × 10 9In some embodiments, the methods described herein, or administration of a G-CSF dimer pharmaceutical composition (e.g., a G-CSF-Fc dimer pharmaceutical composition, such as an abegrastim alfa pharmaceutical composition) after administration of a chemotherapeutic agent to a subject (e.g., at least 24 hours later), reduces ANC from the first appearance of less than about 0.5×10 to less than about 0.5×10 within about 10 days, such as within about 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.2, 0.1 days, or less. 9 / L increases to greater than or equal to about 2.0×10 9 In some embodiments, the methods described herein reduce ANC from less than about 0.5×10-1 to less than about 0.5×10-1 within about 10 days, such as within 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.2, 0.1 days, or less. 9 / L increases to greater than or equal to about 1.5×10 9 / L. In some embodiments, the methods described herein reduce the time to ANC recovery in cycle 1 of chemotherapy for an individual. In some embodiments, the methods described herein reduce the time to ANC recovery in one or more cycles of chemotherapy for an individual (e.g., cycles 1-2 for 2 or more cycles, or cycles 1-3 for 4 or more cycles). In some embodiments, the methods described herein reduce the time to ANC recovery in each cycle of chemotherapy for an individual (e.g., all 2 cycles or all 4 cycles).

[0253] In some embodiments, one or more effects (e.g., therapeutic effects) achieved by the methods described herein or by administering a G-CSF dimer pharmaceutical composition (e.g., a G-CSF-Fc dimer pharmaceutical composition, such as abegrastim alfa pharmaceutical composition) after administration of a chemotherapeutic agent to an individual (e.g., at least 24 hours later) are compared to the effects in an individual not administered a G-CSF dimer drug combination (e.g., any G-CSF dimer pharmaceutical composition described herein). In some embodiments, one or more effects achieved by the methods described herein are compared to the effects achieved by administering a G-CSF dimer pharmaceutical composition (e.g., any G-CSF dimer pharmaceutical composition described herein) to an individual 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). ) or biosimilars, efavirenz (e.g., HM10460A; or ROLVEDON TM ) or biosimilars, or filgrastim In some embodiments, one or more effects achieved by the methods described herein are compared to the effects achieved by administering pegfilgrastim (e.g., ) or biosimilars, efavirenz (e.g. HM10460A; or ROLVEDON TM ) or biosimilars, or filgrastim In some embodiments, the effect of pegfilgrastim (e.g., ) or biosimilars, efavirenz (e.g., HM10460A; or ROLVEDON TM ) or biosimilars, or filgrastim The methods of using the G-CSF dimer pharmaceutical compositions described herein: i) achieve comparable (e.g., within about 5% difference) or better therapeutic effects, and / or ii) have comparable (e.g., about 5% difference) or fewer adverse events, compared to the effects of administering a biosimilar to a subject.

[0254] In some embodiments, the individual is a human. In some embodiments, for each administration, the G-CSF dimer pharmaceutical composition (e.g., G-CSF-Fc dimer pharmaceutical composition, such as abegrastim α pharmaceutical composition) is administered at about 5 mg to about 25 mg, for example, 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, about 18 mg to about 22 mg. In some embodiments, for each administration, 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, or any value therebetween. In some embodiments, for each administration, the G-CSF dimer pharmaceutical composition is administered at about 20 mg. In some embodiments, the individual is a non-human animal. In some embodiments, the G-CSF dimer pharmaceutical composition is administered to a non-human animal at an equivalent human dose. In some embodiments, when the individual is administered 2 or more cycles of a chemotherapeutic agent, the G-CSF dimer pharmaceutical composition is administered in the same amount (e.g., 20 mg) per cycle. In some embodiments, when the individual is administered 2 or more cycles of a chemotherapeutic agent, the G-CSF dimer pharmaceutical composition is administered in different amounts in at least 2 cycles.

[0255] In some embodiments, the individual to be treated or the individual to whom the G-CSF dimer pharmaceutical composition (e.g., G-CSF-Fc dimer pharmaceutical composition, such as abegrastim alfa pharmaceutical composition) is administered meets one or more of the following criteria: i) at least 18 years old for humans; ii) female for individuals with breast cancer (e.g., stage I-III breast cancer); iii) scheduled to receive 2 or more (e.g., 2, 3, 4 or more) cycles of chemotherapy (e.g., neoadjuvant or adjuvant); iv) having an Eastern Cooperative Oncology Group (ECOG) performance status of ≤2; v) having an ANC ≥2.0×10 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 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×ULN); vii) are on contraception for at least one month before 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 abegrastim alfa. In some embodiments, the methods described herein further include selecting an individual who meets one or more of the above criteria.

[0256] In some embodiments, the pharmaceutical composition is administered at a dosage of at least about 0.01 mg / kg, for example, 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 drug combination is administered at a dosage of about 0.01 mg / kg to about 1 mg / kg, for example, at a dosage of 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.

[0257] The pharmaceutical composition can be administered with any desired regimen and frequency. In some embodiments according to any method described herein, the pharmaceutical composition is administered once a day, once every 2 days, once every 3 days, once every 4 days, once every 5 days, once every 6 days, once a week, once every two weeks, once every three weeks, once every four weeks, once every 6 weeks, or once every 8 weeks. In some embodiments, the pharmaceutical composition is administered once every four weeks. In some embodiments, the pharmaceutical composition is administered once every 3 weeks. In some embodiments, the pharmaceutical composition is administered at a dosage of about 20 mg each time, for example, about 20 mg is administered once per chemotherapy cycle. In some embodiments, the method includes administering the pharmaceutical composition in a single dose.

[0258] In some embodiments, the pharmaceutical composition is administered using a syringe (e.g., a prefilled syringe), such as any syringe described herein. In some embodiments, the pharmaceutical composition is administered subcutaneously. In some embodiments, the pharmaceutical composition is contained (e.g., prefilled) in a syringe. 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, such as any G-CSF dimer described herein (e.g., G-CSF-Fc dimer, such as Abegrastim α). In some embodiments, the syringe is sterile. In some embodiments, the syringe is disposable.

[0259] The pharmaceutical composition can be administered to an individual (e.g., a human being, such as an individual suffering from cancer) by various approaches, including, for example, intravenous, intraarterial, intraperitoneal, intravascular, intramuscular, subcutaneous, transmucosal and transdermal. In some embodiments, a sustained continuous release formulation of the pharmaceutical composition can be used. In some embodiments, the implantation of a sustained release device (e.g., a miniature osmotic pump) can be used. Other delivery methods include, but are not limited to, the use of liposome preparations 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 by intramuscular injection. In some embodiments, the pharmaceutical composition is administered subcutaneously. When administered subcutaneously, such pharmaceutical compositions are generally administered with a volume of less than about 2.0 mL (e.g., about 1.5 mL, about 1 mL, or about 0.5 mL) per injection site. In some embodiments, the injection volume is 1 mL.

[0260] Neutropenia

[0261] Neutropenia is characterized by a peripheral blood neutrophil count of less than 1.8 × 10 9 / L and children below 1.5×10 9 / L. Neutropenia is often a precursor to infection: the lower the neutrophil count, the higher the risk of infection.

[0262] Guidelines for classifying neutropenia are shown in Table A. The frequency and severity of infections due to neutropenia are also influenced by other factors (eg, integrity of mucous membranes and skin, function and levels of immunoglobulins, lymphocytes, monocytes, complement system, etc.).

[0263] Table A. Classification of Neutropenia

[0264] Neutropenia Neutrophil count Risk of infection slight <![CDATA[1.0×10 9 / L to 1.8×10 9 / L]]> Minimum Moderate <![CDATA[0.5×10 9 / L to 1.0×10 9 / L]]> Increased serious <![CDATA[<0.5×10 9 / L]]> serious

[0265] According to the cause of neutropenia, common clinical neutropenia can be divided into the following categories: hematopoietic system production disorders caused by secondary factors such as drugs, radiation, chemical agents and infections; changes in distribution and circulation in the body, increased utilization and turnover. The severity of chemotherapy-induced neutropenia in tumor patients usually depends on the dose of chemotherapy, and repeated use of chemotherapy may have a cumulative effect on neutropenia. The main clinical consequence of neutropenia is infectious complications. Most infections in these patients are mainly caused by aerobic bacteria, including Gram-negative bacteria (Escherichia coli, Klebsiellapheumoniae and Pseudomonas aeruginosa), Gram-positive bacteria (Staphylococci, α-hemolytic Streptococci and Staphylococcus aureus) and fungi.

[0266] Cytotoxic chemotherapy remains one of the main treatments for cancer. The biggest disadvantage of chemotherapy is that it indiscriminately kills rapidly proliferating and differentiating healthy cells as well as tumor cells. The toxicity caused by chemotherapy is mainly reflected in the hematopoietic system, which is clinically known as chemotherapy-induced neutropenia.

[0267] Neutropenia may delay the next treatment cycle, which directly affects the therapeutic effect of chemotherapy. Severe neutropenia, that is, ANC less than 0.5×10 9 / L, which can lead to infection, organ failure, and even threaten the patient's life.

[0268] 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).

[0269] Thus, in some embodiments, a method for treating or preventing neutropenia in an individual (e.g., a human, such as a human with cancer) in need thereof is provided, the method comprising administering to the individual an effective amount of any pharmaceutical composition described herein (e.g., a G-CSF-Fc dimer pharmaceutical composition, such as an abegrastim alfa pharmaceutical composition) after (e.g., at least 24 hours after) administration of an agent that induces neutropenia (e.g., a chemotherapeutic agent that can induce neutropenia).

[0270] Diseases treated with chemotherapeutic agents

[0271] In some embodiments, a chemotherapeutic agent is administered to an individual to treat cancer.

[0272] Treatable cancers 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 adenocarcinoma, ampullary cancer, esophageal cancer, kidney cancer, thyroid cancer, squamous cell carcinoma, lung cancer, B-cell lymphoma, acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), non-Hodgkin lymphoma (NHL), Burkitt lymphoma, Wilms' tumor, etc. In some embodiments, cancer is a solid cancer. In some embodiments, cancer is a liquid cancer.

[0273] In some embodiments, cancer is non-myeloid cancer, such as non-myeloid malignancies. In some embodiments, cancer is selected from breast cancer, non-small cell lung cancer, small cell lung cancer, ovarian cancer, sarcoma, urothelial carcinoma, germ cell tumors and non-Hodgkin lymphoma. In some embodiments, cancer is breast cancer. In some embodiments, cancer is stage I-III or stage II-IV breast cancer. In some embodiments, breast cancer is metastatic breast cancer. In some embodiments, breast cancer is non-metastatic breast cancer.

[0274] In some embodiments, the methods of administering the pharmaceutical compositions of G-CSF dimers described herein ensure that the individual does not delay the treatment regimen of the chemotherapeutic agent, or ensures that the delay in the treatment regimen of the chemotherapeutic agent does not exceed about 5 days, such as no more than about any of 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 the chemotherapeutic agent is to be administered for 2 or more cycles, the methods of administering the pharmaceutical compositions of G-CSF dimers described herein ensure that the individual does not delay the treatment regimen of the chemotherapeutic agent in each cycle, or the delay is only in cycle 2 (e.g., no more than about 5 days), or the delay is not in all cycles (e.g., only in cycles 2 and 3 out of 4 cycles).

[0275] Chemotherapeutic agents

[0276] In some embodiments, the chemotherapeutic agent is a myelosuppressive chemotherapeutic agent. In some embodiments, the myelosuppressive chemotherapeutic agent is selected from epirubicin, docetaxel, cyclophosphamide, doxorubicin, etoposide, cisplatin, paclitaxel, topotecan, vincristine, methylprednisolone, cytarabine, and combinations thereof.

[0277] In some embodiments, the cancer to be treated by the chemotherapeutic agent is breast cancer. In some embodiments, two or more chemotherapeutic agents are administered to an individual, including: i) epirubicin and cyclophosphamide (e.g., epirubicin 100 mg / m 2 and 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. 75 mg / m 2 Docetaxel and 60 mg / m 2 In some embodiments, two or more chemotherapeutic agents are administered simultaneously in the same formulation or in separate formulations. In some embodiments, two or more chemotherapeutic agents are administered sequentially.

[0278] In some embodiments, the chemotherapeutic agent induces neutropenia (e.g., any grade of neutropenia), such as grade 4 neutropenia. In some embodiments, the chemotherapeutic agent induces FN. In some embodiments, the chemotherapeutic agent reduces ANC to less than about 0.5×10 9 / L. In some embodiments, the chemotherapeutic agent reduces ANC, for example, reduces ANC by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more. In some embodiments, the duration of neutropenia (DN) induced by the chemotherapeutic agent is at least about 2 days, for example, at least about 3, 4, 5, 6, 7, 8, 9, 10 days or more.

[0279] VII. Systems, kits and preparation methods

[0280] Also provided are systems, kits, and methods of making the pharmaceutical compositions described herein (eg, abegrastim alpha pharmaceutical compositions).

[0281] In some embodiments, a lyophilized pharmaceutical composition is provided. WO97 / 04801 describes a lyophilized pharmaceutical composition suitable for subcutaneous administration. This lyophilized pharmaceutical composition can be reconstituted to a high protein concentration with a suitable diluent, and the reconstituted formulation can be subcutaneously administered to an individual to be imaged, diagnosed or treated herein.

[0282] The pharmaceutical composition for in vivo administration must be sterile. This is easily achieved by, for example, filtration through a sterile filtration membrane. In some embodiments, a syringe containing any one of the pharmaceutical compositions described herein (e.g., a pharmaceutical composition of Abegrastim α) is provided. In some embodiments, the syringe is sterile. In some embodiments, the syringe is disposable. In some embodiments, the syringe is pre-filled with about 1 mL of the pharmaceutical composition described herein.

[0283] Also provided are kits comprising any of the pharmaceutical compositions described herein (eg, abegrastim alpha pharmaceutical composition). The kits can be used in any of the methods of treating or preventing a disease or condition described herein.

[0284] In some embodiments, a kit comprising a G-CSF dimer (e.g., a G-CSF-Fc dimer, such as abegrastim alpha) is provided. In some embodiments, the kit further comprises other components required for preparing a G-CSF dimer pharmaceutical composition (e.g., any pharmaceutical composition described herein).

[0285] In some embodiments, the kit further comprises a device capable of delivering the pharmaceutical composition to an individual.One device useful for applications such as parenteral delivery is a syringe for injecting the pharmaceutical composition into a subject.

[0286] In some embodiments, the kit further comprises one or more therapeutic agents for treating a disease or disorder (eg, cancer). In some embodiments, the therapeutic agent is a chemotherapeutic agent.

[0287] The kit of the present application can be in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed polyester film or plastic bags), etc. The kit can optionally provide additional components, such as buffer and explanatory information.

[0288] The application also provides articles. Articles can include containers and labels or package inserts on or associated with containers. Suitable containers include vials (such as sealed vials), bottles, jars, soft packages, etc. Typically, the container holds the composition and can have a sterile access port (for example, the container can be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle). The label or package insert indicates that the composition is used for imaging, diagnosing or treating a specific condition of an individual. The label or package insert will also include instructions for administering the composition to an individual and / or for imaging an individual. The label can indicate reconstruction and / or use instructions. The container holding the composition can be a multi-purpose bottle that allows repeated administration (for example, 2-6 administrations) of the reconstructed preparation. The package insert refers to instructions typically contained in a diagnostic or therapeutic product commercial package, wherein the instructions contain information about the indications, usage, dosage, administration, contraindications and / or warnings for using such diagnostic products. In addition, the article of manufacture may further include 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 also include other materials desirable from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

[0289] The kit or article of manufacture may include multiple unit doses of the composition packaged in quantities sufficient for storage and use in pharmacies (eg, hospital pharmacies and compounding pharmacies) and instructions for use.

[0290] In some embodiments, a method of preparing any pharmaceutical composition described herein is provided, comprising one or more (e.g., all) of the following steps: (i) culturing host cells under conditions suitable for expressing a G-CSF dimer (e.g., any G-CSF dimer described herein, such as abegrastim α); (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 the following: affinity chromatography, viral inactivation, ion exchange chromatography, mixed mode chromatography, and filtration. In some embodiments, a method of preparing any pharmaceutical composition described herein is provided, comprising formulating the purified G-CSF dimer with a buffer, stabilizer, tonicity agent, and surfactant as 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 Example 7 and Table 13 for exemplary methods.

[0291] 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 G-CSF dimers. In some embodiments, the nucleic acid encoding the G-CSF dimer comprises the sequence of SEQ ID NO:11.

[0292] Those skilled in the art will recognize that some embodiments are possible within the scope and spirit of the present invention. The present invention will now be described in more detail with reference to the following non-limiting examples. The following examples further illustrate the present invention, but certainly should not be construed as limiting its scope in any way.

[0293] VIII. Exemplary Embodiments

[0294] 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 a G-CSF dimer, wherein the G-CSF dimer comprises two monomer subunits, each monomer subunit 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, comprising (or consisting essentially of, or consisting of): a) about 1 mg / mL to about 100 mg / mL of a G-CSF dimer, wherein the G-CSF dimer comprises two monomer subunits, and wherein each monomer subunit comprises a G-CSF monomer (e.g., SEQ ID NO: 1), an Fc fragment (e.g., any one of SEQ ID NOs: 2-5), and an optional linker connecting the G-CSF monomer and the Fc fragment (e.g., SEQ ID NO: 12); (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 a G-CSF dimer, wherein the G-CSF dimer comprises two monomer subunits, each monomer subunit 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 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 a G-CSF dimer, wherein the G-CSF dimer is abergolistim alpha; (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 a G-CSF dimer, wherein the G-CSF dimer comprises two monomer subunits, each monomer subunit 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 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 a G-CSF dimer, wherein the G-CSF dimer comprises two monomer subunits, each monomer subunit 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 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 a G-CSF dimer, wherein the G-CSF dimer comprises two monomer subunits, each monomer subunit 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 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 a G-CSF dimer, wherein the G-CSF dimer comprises two monomer subunits, each monomer subunit 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 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, the concentration of G-CSF dimer is from about 5 mg / mL to about 50 mg / mL, for example, any one of 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 G-CSF dimer is from about 18 mg / mL to about 22 mg / mL, for example, about 20 mg / mL. In some embodiments, the buffer is sodium acetate. In some embodiments, the concentration of sodium acetate is from about 1 mM to about 30 mM, for example, about 10 mM. In some embodiments, the stabilizer is EDTA. In some embodiments, the concentration of EDTA is from about 0.1 mM to about 10 mM, for example, any one of 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 tonicity agent is sorbitol. In some embodiments, the pharmaceutical composition comprises about 1% (w / v) to about 8% (w / v) of sorbitol, for example, about 1% (w / v), about 2% (w / v), about 3% (w / v), about 4% (w / v), about 5% (w / v), about 6% (w / v), about 7% (w / v) or about 8% (w / v) of any one of sorbitol. In some embodiments, the pharmaceutical composition comprises about 5% (w / v) of 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) of sucrose, for example, about 9% (w / v) of sucrose. In some embodiments, the surfactant is selected from polysorbate, poloxamer, polyoxyethylene alkyl ether, alkylphenyl polyoxyethylene ether and combinations thereof. In some embodiments, the surfactant is a nonionic surfactant. 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, for example, about 0.006% (w / v), about 0.007% (w / v), about 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) of any one of PS20. In some embodiments, the pharmaceutical composition comprises less than about 0.03% (w / v) PS20. In some embodiments, the pharmaceutical composition comprises about 0.01% (w / v) PS20. In some embodiments, 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. 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 91%, 92%, 93%, 94%, 95%, 99% or more) as measured by SE-HPLC. In some embodiments, the pharmaceutical composition comprises no more than about 2% aggregates. In some embodiments, the osmolarity of the pharmaceutical composition is about 310 mOsm / kg. In some embodiments, after storage at about 2°C to about 8°C for at least about 1 month (e.g., at least about 2 months, such as about 6 months, 12 months, 24 months, 36 months or more), the pharmaceutical composition comprises at least about 96% of G-CSF dimer as measured by SE-HPLC. In some embodiments, the pharmaceutical composition comprises at least about 96% of G-CSF dimer as measured by SE-HPLC after storage for up to about 36 months at about 2°C to about 8°C. In some embodiments, the pharmaceutical composition comprises at least about 95.5% of G-CSF dimer as measured by rCE-SDS after storage for at least about 1 month (e.g., at least about 2 months, such as any one of about 6 months, 12 months, 24 months, 36 months, or longer) at about 2°C to about 8°C. In some embodiments, the pharmaceutical composition comprises at least about 95.5% of G-CSF dimer as measured by rCE-SDS after storage for up to about 36 months at about 2°C to about 8°C. In some embodiments, the pharmaceutical composition comprises at least about 94% of G-CSF dimer as measured by nrCE-SDS after storage for at least about 1 month (e.g., at least about 2 months, such as any one of about 6 months, 12 months, 24 months, 36 months, or longer) 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 storage for up to about 36 months at about 2°C to about 8°C. In some embodiments, the pharmaceutical composition comprises no more than about 1.5% HMWS as measured by rCE-SDS after storage for at least about 1 month (e.g., at least about 2 months, such as any one of about 6 months, 12 months, 24 months, 36 months, or longer) at about 2°C to about 8°C. In some embodiments, the pharmaceutical composition comprises no more than about 1.5% HMWS as measured by rCE-SDS after storage for up to about 36 months at about 2°C to about 8°C. In some embodiments, the pharmaceutical composition comprises no more than about 1.5% HMWS as measured by nrCE-SDS after storage for at least about 1 month (e.g., at least about 2 months, such as any one of about 6 months, 12 months, 24 months, 36 months, or longer) at about 2°C to about 8°C. In some embodiments, after storage at about 2°C to about 8°C for up to about 36 months, the pharmaceutical composition comprises no more than about 1.5% HMWS as measured by nrCE-SDS. In some embodiments, after storage at about 2°C to about 8°C for at least about 1 month (e....

Claims

1. A pharmaceutical composition comprising: (a) about 1 mg / mL to about 100 mg / mL of granulocyte colony stimulating factor (G-CSF) dimer; (b) about 1 mM to about 50 mM buffer; (c) about 0.1 mM to about 20 mM of a stabilizer; (d) from about 1% (w / v) to about 15% (w / v) of a tonicity agent; and (e) from about 0.001% (w / v) to about 0.1% (w / v) of a surfactant.

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

3. The pharmaceutical composition according to claim 2, wherein the G-CSF monomer comprises the sequence of SEQ ID NO:

1.

4. The pharmaceutical composition according to claim 2 or 3, wherein the G-CSF monomer is linked to the dimerization domain via an optional linker.

5. The pharmaceutical composition according to claim 4, wherein the linker: (i) is about 6 to about 30 amino acids in length; and / or (ii) comprising the amino acid sequence of SEQ ID NO:

12.

6. The pharmaceutical composition according to any one of claims 2 to 5, wherein the dimerization domain comprises at least two cysteines capable of forming intermolecular disulfide bonds.

7. The pharmaceutical composition according to any one of claims 2-6, wherein the dimerization domain comprises at least a portion of an Fc fragment.

8. The pharmaceutical composition according to claim 7, wherein the Fc fragment: (i) comprises a CH2 and a CH3 domain; and / or (ii) derived from IgG1 Fc, IgG2 Fc, IgG4 Fc or fragments or variants thereof.

9. The pharmaceutical composition according to claim 7 or 8, wherein the Fc fragment comprises an amino acid sequence selected from SEQ ID NO: 2-5.

10. The pharmaceutical composition of any one of claims 2-9, wherein the G-CSF monomer is N-terminal relative to the dimerization domain within each monomer subunit.

11. The pharmaceutical composition of any one of claims 2-10, wherein each monomer subunit comprises an amino acid sequence of any one of SEQ ID NOs: 6-10 or a variant thereof having at least about 90% sequence identity with an amino acid sequence of any one of SEQ ID NOs: 6-10.

12. A pharmaceutical composition according to any one of claims 2-11, wherein each monomer subunit comprises the amino acid sequence of SEQ ID NO:

6.

13. The pharmaceutical composition according to any one of claims 1 to 12, wherein the G-CSF dimer is abegrastim alfa.

14. The pharmaceutical composition of any one of claims 1-13, wherein the concentration of the G-CSF dimer is about 5 mg / mL to about 50 mg / mL.

15. The pharmaceutical composition of any one of claims 1-14, wherein the concentration of the G-CSF dimer is about 20 mg / mL.

16. The pharmaceutical composition according to any one of claims 1-15, wherein the buffer is sodium acetate.

17. The pharmaceutical composition of claim 16, wherein the concentration of sodium acetate is about 1 mM to about 30 mM.

18. The pharmaceutical composition of claim 16 or 17, wherein the concentration of sodium acetate is about 10 mM.

19. The pharmaceutical composition according to any one of claims 1-18, wherein the stabilizer is EDTA.

20. The pharmaceutical composition of claim 19, wherein the concentration of EDTA is about 0.1 mM to about 10 mM.

21. The pharmaceutical composition of claim 19 or 20, wherein the concentration of EDTA is about 1 mM.

22. The pharmaceutical composition of any one of claims 1-21, wherein the tonicity agent is sorbitol.

23. The pharmaceutical composition of claim 22, wherein the pharmaceutical composition comprises about 1% (w / v) to about 8% (w / v) sorbitol.

24. The pharmaceutical composition of claim 22 or 23, wherein the pharmaceutical composition comprises about 5% (w / v) sorbitol.

25. The pharmaceutical composition of any one of claims 1-21, wherein the tonicity agent is sucrose.

26. The pharmaceutical composition of claim 25, wherein the pharmaceutical composition comprises about 5% (w / v) to about 15% (w / v) sucrose.

27. The pharmaceutical composition of claim 25 or 26, wherein the pharmaceutical composition comprises about 9% (w / v) sucrose.

28. The pharmaceutical composition according to any one of claims 1-27, wherein the surfactant is selected from the group consisting of polysorbates, poloxamers, polyoxyethylene alkyl ethers, alkylphenyl polyoxyethylene ethers, and combinations thereof.

29. The pharmaceutical composition of any one of claims 1-28, wherein the surfactant is polysorbate 20 (PS20) or polysorbate 80 (PS80).

30. The pharmaceutical composition of claim 29, wherein the pharmaceutical composition comprises about 0.005% (w / v) to about 0.05% (w / v) PS20.

31. The pharmaceutical composition of claim 29 or 30, wherein the pharmaceutical composition comprises about 0.01% (w / v) PS20.

32. The pharmaceutical composition of any one of claims 1-31, wherein the pH of the pharmaceutical composition is about 4.2 to about 6.

2.

33. The pharmaceutical composition of any one of claims 1-32, wherein the pH of the pharmaceutical composition is about 4.8 to about 5.

8.

34. The pharmaceutical composition of any one of claims 1-33, wherein the pH of the pharmaceutical composition is about 5.0 to about 5.

4.

35. The pharmaceutical composition of any one of claims 1-34, wherein the pH of the pharmaceutical composition is about 5.

2.

36. A pharmaceutical composition according to any one of claims 1-24 and 28-35, comprising: (a) about 20 mg / mL of 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, The pH of the pharmaceutical composition is about 5.

2.

37. The pharmaceutical composition of any one of claims 1-36, wherein the pharmaceutical composition is contained in a syringe.

38. The pharmaceutical composition of claim 37, wherein the volume of the pharmaceutical composition within the syringe is about 1 mL.

39. The pharmaceutical composition according to claim 37 or 38, wherein the syringe is for single use.

40. The pharmaceutical composition of any one of claims 37-39, wherein the syringe is sterile.

41. A method of treating or preventing a disease or condition in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition according to any one of claims 1-40.

42. The method of claim 41, wherein the pharmaceutical composition is administered by: (i) a dose of about 0.01 mg / kg to about 1 mg / kg; and / or (ii) Once every three weeks.

43. The method of claim 41 or 42, wherein the disease or condition is selected from neutropenia, stroke, spinal cord injury, neurological diseases 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 to peripheral blood in allogeneic hematopoietic stem cell transplantation.

44. The method of any one of claims 41-43, wherein the disease or condition is neutropenia.

45. The method of claim 44, wherein the neutropenia is chemotherapy-induced neutropenia or radiation therapy-induced neutropenia.

46. ​​The method of claim 45, wherein the method comprises administering to the individual an effective amount of the pharmaceutical composition after administering to the individual a chemotherapeutic agent.

47. The method of claim 46, wherein the chemotherapeutic agent is a myelosuppressive chemotherapeutic agent.

48. The method of claim 46 or 47, wherein the individual is administered at least 4 cycles of a chemotherapeutic agent, and wherein the pharmaceutical composition is administered after administration of the chemotherapeutic agent in each of the at least 4 cycles.

49. The method of any one of claims 41-48, wherein the pharmaceutical composition is administered at about 5 mg to about 25 mg of the G-CSF dimer per administration.

50. The method of any one of claims 41-49, wherein the pharmaceutical composition is administered subcutaneously.

51. The method of any one of claims 41-50, wherein the individual is a human.

52. A method for preparing a pharmaceutical composition according to any one of claims 1-40, comprising: (i) culturing a host cell under conditions suitable for expressing the 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 the buffer, the stabilizer, the tonicity agent and the surfactant.

53. The method of claim 52, wherein the purification step comprises one or more of the following: affinity chromatography, viral inactivation, ion exchange chromatography, mixed mode chromatography, and filtration.

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