G CSF Human, CHO
Description
Biological Activity and Potency
G-CSF Human, CHO demonstrates robust biological activity:
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In Vitro Potency: ED₅₀ < 0.1 ng/ml in murine M-NFS-60 cell proliferation assays, equivalent to >1 × 10⁷ units/mg .
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Functional Effects:
Manufacturing and Standardization
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Production System: Expressed in CHO cells for proper glycosylation and folding, ensuring bioactivity comparable to natural G-CSF .
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International Standards:
Pharmacokinetic Properties
| Parameter | CHO-Derived G-CSF | E. coli-Derived G-CSF |
|---|---|---|
| Half-Life | 3.5–4.5 hours | 2–3 hours |
| Clearance | Saturable, receptor-mediated | Renal-dominated |
| Bioavailability | ~60% (subcutaneous) | ~60% (subcutaneous) |
CHO-derived G-CSF exhibits prolonged activity due to glycosylation, reducing dosing frequency compared to E. coli variants .
Chemotherapy-Induced Neutropenia (CIN)
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Efficacy: Reduces severe neutropenia incidence by 50% and febrile neutropenia by 30–50% in cancer patients .
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Dosing: 5 μg/kg/day subcutaneously or intravenously until neutrophil recovery .
Hematopoietic Stem Cell Transplantation
Other Applications
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Chronic Neutropenia: Restores neutrophil counts to >1.5 × 10⁹/L in 90% of patients .
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Reproductive Medicine: Improves implantation rates in recurrent implantation failure (66% vs. 30% with HCG) .
Comparative Advantages Over Non-Glycosylated Forms
| Feature | CHO-Derived G-CSF | E. coli-Derived G-CSF |
|---|---|---|
| Glycosylation | O-linked (Thr-133) | None |
| Immunogenicity | Lower | Higher |
| In Vivo Stability | Enhanced | Reduced |
| Regulatory Status | WHO-compliant | WHO-compliant |
Research Advancements
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Next-Generation Variants: Fc-fusion proteins (e.g., G-CSF-IgG4) extend half-life to 15–24 hours via reduced renal clearance .
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Safety Profile: No increased leukemia risk observed in long-term studies .
Challenges and Future Directions
Product Specs
Q&A
How can researchers confirm the structural integrity of CHO-expressed G-CSF compared to native human G-CSF?
To validate structural equivalence, researchers should perform tandem mass spectrometry (MS/MS) for amino acid sequencing and disulfide bond mapping. Natural and CHO-expressed G-CSF both contain 174 amino acids with identical sequences, including free Cys-17 and two intramolecular disulfide bonds (Cys-36–Cys-42 and Cys-64–Cys-74) . Circular dichroism (CD) spectroscopy can confirm secondary structure similarity, as both forms exhibit overlapping spectra . Additionally, O-glycosylation at Thr-133 should be verified using β-elimination followed by chromatographic analysis .
Key Structural Validation Data
What experimental design ensures accurate measurement of G-CSF bioactivity in vitro?
Use a standardized cell proliferation assay with murine M-NFS-60 cells, which exhibit dose-dependent responsiveness to G-CSF. The ED₅₀ should be <0.1 ng/mL, corresponding to a specific activity of >1×10⁷ units/mg . Include the following controls:
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Negative control: Culture medium without G-CSF.
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Reference standard: WHO International Standard for G-CSF.
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Dose-response curve: 0.01–10 ng/mL range with triplicate measurements.
Neutralizing antibodies against G-CSF receptor (G-CSFR) can confirm specificity .
How does glycosylation in CHO cells impact G-CSF function?
CHO cells confer O-glycosylation at Thr-133, which does not alter receptor binding or in vitro activity but may influence pharmacokinetics. To assess functional relevance:
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Compare deglycosylated (e.g., PNGase F-treated) and native G-CSF in serum half-life studies.
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Use surface plasmon resonance (SPR) to measure binding kinetics to G-CSFR .
Data indicate identical in vivo activity between glycosylated and nonglycosylated forms in murine models .
How can contradictory data on G-CSF-induced neurogenesis in Alzheimer’s disease models be resolved?
Conflicting results may arise from differences in Aβ aggregate localization or administration protocols. To address this:
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Model standardization: Use APP/PS1 transgenic mice with defined Aβ plaque distribution .
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Dose optimization: Subcutaneous 250 µg/kg/day for 5 days showed cognitive rescue .
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Mechanistic validation: Track BrdU⁺ cells in hippocampal and cortical regions via immunohistochemistry .
Neurogenic Effects of G-CSF in AD Models
| Parameter | G-CSF-Treated Group | Control Group | p-value |
|---|---|---|---|
| BrdU⁺ cells/mm² (Cortex) | 12.4 ± 1.8 | 3.1 ± 0.9 | <0.001 |
| Morris water maze latency | 28.2 s ± 4.1 | 52.7 s ± 6.3 | <0.01 |
What methodologies identify soluble G-CSF receptors in physiological conditions?
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Native PAGE with biotinylated G-CSF: Incubate human serum with biotinylated G-CSF, resolve via non-denaturing PAGE, and detect using streptavidin-peroxidase. This reveals 75 kDa and 85 kDa soluble receptors .
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RT-PCR for receptor isoforms: Use primers spanning the transmembrane domain to distinguish full-length (type I) and truncated (type II) G-CSFR mRNA .
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Functional assays: Add soluble receptors to bone marrow cultures; >50% suppression of granulocyte colony formation indicates biological relevance .
How does G-CSF upregulate E-selectin ligands on myeloid cells, and what are the implications for experimental design?
G-CSF induces HCELL (CD44 glycovariant) and a novel 65 kDa ligand via increased ST3Gal-IV, FucT-IV, and FucT-VII expression . To study this:
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Flow adhesion assays: Use TNF-α-activated HUVECs under shear stress (1–2 dyn/cm²).
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Enzymatic inhibition: Treat cells with neuraminidase (sialidase) or fucosidase to abolish binding .
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Transcript analysis: Quantify glycosyltransferase mRNA via qRT-PCR .
G-CSF-Induced E-Selectin Ligand Expression
| Ligand Type | Baseline MFI | Post-G-CSF MFI | Fold Change |
|---|---|---|---|
| HCELL (CD44) | 120 ± 15 | 450 ± 40 | 3.75 |
| 65 kDa ligand | 80 ± 10 | 300 ± 25 | 3.75 |
What strategies optimize CHO expression vectors for high-yield G-CSF production?
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Codon optimization: Replace rare codons (e.g., AGA for arginine) with CHO-preferred CGT .
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Promoter engineering: Use a hybrid CMV-IE/HTLV promoter for enhanced transcription .
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Fed-batch culture: Maintain glucose at 2–4 g/L and dissolved oxygen at 30% saturation .
This approach achieves titers >1 g/L with >95% monomeric purity after hydrophobic interaction chromatography .
How should researchers address batch-to-batch variability in disulfide bond formation?
Implement a two-tiered QC protocol:
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Reductive alkylation: Compare reduced vs. non-reduced SDS-PAGE to detect free thiols .
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Peptide mapping: Use tryptic digestion followed by LC-MS/MS to confirm Cys-36–Cys-42 and Cys-64–Cys-74 linkages .
Re-calibrate bioreactor redox potential if >5% of molecules exhibit incorrect disulfide pairing .
Methodological Recommendations
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For in vivo studies: Use 6–8-week-old C57BL/6 mice for consistency with published pharmacokinetic models .
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In endotoxin testing: Apply the Limulus amebocyte lysate (LAL) assay with a threshold of <0.1 EU/µg .
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Data contradiction analysis: Cross-validate using orthogonal methods (e.g., SPR alongside cell-based assays) .
Product Science Overview
Granulocyte-Colony Stimulating Factor (G-CSF) is a glycoprotein that plays a crucial role in hematopoiesis, the process by which blood cells are formed. It specifically stimulates the bone marrow to produce granulocytes and stem cells and release them into the bloodstream. The recombinant form of G-CSF, produced using Chinese Hamster Ovary (CHO) cells, is widely used in clinical settings to treat neutropenia, a condition characterized by an abnormally low number of neutrophils, which are a type of white blood cell essential for fighting infections.
The discovery of G-CSF dates back to the early 1980s. It was first isolated from human cells by Malcolm Moore and Karl Welte in 1984 . The human form of G-CSF was cloned by research groups from Japan and Germany/United States in 1986 . This breakthrough led to the development of recombinant G-CSF, which has since become a cornerstone in the treatment of various conditions, particularly those related to cancer therapy.
Chinese Hamster Ovary (CHO) cells are a type of cell line derived from the ovary of the Chinese hamster. These cells are commonly used in biological and medical research due to their ability to produce large quantities of recombinant proteins. The production of recombinant G-CSF involves inserting the human G-CSF gene into CHO cells, which then express the protein. This method ensures a high yield and purity of the recombinant protein, making it suitable for therapeutic use.
Recombinant G-CSF is primarily used to treat neutropenia in patients undergoing chemotherapy or bone marrow transplantation. It helps to reduce the risk of infections by increasing the number of neutrophils in the blood . Additionally, G-CSF is used in the mobilization of hematopoietic stem cells for collection and subsequent transplantation. It has also shown promise in improving ovum quality and maturity in women with poor ovarian response undergoing in vitro fertilization (IVF) .
G-CSF binds to specific receptors on the surface of hematopoietic stem cells and granulocyte precursors in the bone marrow. This binding activates intracellular signaling pathways that promote the proliferation, differentiation, and survival of these cells. As a result, there is an increase in the production and release of neutrophils into the bloodstream .
The safety and efficacy of recombinant G-CSF have been well-documented in numerous clinical trials. It has been shown to significantly reduce the incidence of febrile neutropenia and other complications associated with low neutrophil counts in cancer patients . The use of pegylated forms of G-CSF, which have a longer half-life, has further improved patient outcomes by reducing the frequency of administration .
Research into G-CSF continues to evolve, with ongoing studies exploring its potential applications in other medical conditions and its role in enhancing the efficacy of existing treatments. Advances in biotechnology may also lead to the development of more efficient and cost-effective methods for producing recombinant G-CSF.
