Validation of an in vitro biological method for filgrastim potency assessment

INTRODUCTION. Filgrastim is a human granulocyte colony-stimulating factor produced using recombinant DNA technology. Since filgrastim quality control necessitates a validated method, establishing the entire method validation parameters is of high priority; confirming the previously established parameters gains primary importance when substituting critical reagents/ materials/parameters.

AIM. This study aimed to select and confirm a number of validation parameters for in vitro biological method that would assess filgrastim potency using NFS-60 cell line.

MATERIALS AND METHODS. Filgrastim potency was assessed by fluorescence intensity of alamarBlue™ reagent at excitation wavelengths of 530 nm and emission of 620 nm directly proportional to proliferation level of NFS-60 cells exposed to filgrastim 0.1 to 208 IU/mL. Statistical analysis of the results was performed using the 4-parameter logistic function 4PL and the parallel line analysis in PLA 2.0.0 software.

RESULTS. The method was deemed precise: the coefficient of variation (CV) in the repeatability study was 6.20%, and the intermediate precision study showed CV 1.19% (acceptance criterion CV≤25%). The method was linear; the coefficient of determination for the linear function was R2=0.99 (acceptance criterion R2≥0.95). The degree of recovery was 101.6% and did not exceed ±10% of the expected value. The method was robust to an increased density of the cell suspension from 1.5×105 to 3.0×105 cells/mL (CV=0.07%) and to prolonged incubation of filgrastim samples with the cell suspension (from 48 to 72 h, CV=4.2%) or with fluorescent dye (from 4 to 6 h, CV=2.05%).

CONCLUSIONS. The in vitro method assessing specific potency of filgrastim-based preparations using NFS-60 cell line is linear, precise, and accurate, and has proven to be stable under controlled changes of certain parameters. Confirmed applicability of NFS-60 cell line for assessing the potency of filgrastim-based preparations is essential for manufacturers, since it expands the range of cell cultures used for quality assessment of preparations.

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