In vitro bioassay with enhanced sensitivity for human granulocyte colony-stimulating factor

Area of Science:

• Biotechnology
• Cell Biology
• Pharmacology

Background:

• Human granulocyte colony-stimulating factor (hG-CSF) is crucial for hematopoiesis.
• Accurate quantification of hG-CSF activity is essential for pharmaceutical development.
• Existing methods may lack the required sensitivity or specificity for potency assays.

Purpose of the Study:

• To develop and validate a sensitive cell proliferation-based assay for human granulocyte colony-stimulating factor (hG-CSF) activity.
• To establish optimal conditions for quantitative analysis of hG-CSF.
• To assess the suitability of the assay for pharmaceutical potency determination.

Main Methods:

• Utilized a murine myeloid leukemia cell line (NFS-60) for enhanced hG-CSF sensitivity.
• Employed a colorimetric microassay measuring formazan production via MTT reduction.
• Implemented a five-dose parallel line test design for potency determination, adhering to pharmacopoeial standards.

Main Results:

• Established an optimal quantitative analysis range of 4-60 pg/mL for hG-CSF.
• Demonstrated high assay validity with inter-assay RSD between 5.2-12.0% and potencies within 90-110% error limits.
• Confirmed assay specificity and identified pH-dependent activity variations for unglycosylated rhG-CSF.

Conclusions:

• The developed MTT-based colorimetric microassay provides a sensitive and reliable method for hG-CSF activity determination.
• The assay meets pharmacopoeial requirements for potency assessment of pharmaceutical hG-CSF formulations.
• Further investigation into pH effects on rhG-CSF activity is warranted for specific formulations.