Analytical Method Validation for Testing of Limit of High Molecular Weight Proteins in Filgrastim Biopharmaceutical Products


  • Soraya Hengsawas Surasarang Bureau of Drug and Narcotic, Department of Medical Sciences, Ministry of Public Health, Thailand
  • Chanida Karnpracha
  • Boontarika Boonyapiwat


filgrastim, high molecular weight proteins, aggregation, method validation, size exclusion chromatography


Filgrastim is a biopharmaceutical drug used for treatment chemotherapy-induced neutropenia in cancer patients. High molecular weight proteins (HMWP) of filgrastim can lead to loss of efficacy and immunogenicity. Limit of HMWP is one of the test items showing quality of filgrastim products. However, a compendial method used for determination of these impurities in pharmaceutical products has not yet been available. In this study, a size-exclusion chromatographic (SE-HPLC) method was validated for determination of HMWP of filgrastim. Method validation conducted parameters including specificity, limit of quantitation (LOQ) and precision. The results showed that the analysis of HMWP of filgrastim was not interfered by the excipients and other reagents in the analytical system. The method was sensitive with the lowest limit of quantitation of 0.0002 mg/mL. In addition, the percent relative standard deviation of repeatability and intermediate precision was in a range of 1.9-3.5% and 1.9-7.3%, respectively. These values were within the limits calculated from Horwitz’s equation. Thus, the size-exclusion chromatography (SEC) method was specific, sensitive, precise and valid for determination of HMWP of filgrastim in pharmaceutical products. This validated method has been utilized as a standard method to quantify the HMWP of filgrastim in biopharmaceutical products collected from all over nation by the Thai FDA.


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