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Validation of an Automated Fluorescence- and Image-Based Viable Cell Counting Method for Fecal Microbiota Transplantation Drug Products
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DOI：10.1002/biot.70269.png)
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Abstract
En 中文
Fecal microbiota transplantation (FMT) is an established treatment for recurrent Clostridioides difficile infection. As a drug product, FMT is subject to pharmaceutical quality standards, including accurate quantification of viable bacteria to ensure product consistency. We developed and validated an automated fluorescence- and image-based viable-cell counting method for FMT products; total-cell enumeration was examined secondarily. The QUANTOM Tx system (Logos Biosystems) was validated according to ICH Q2(R2) using Escherichia coli and Bacillus subtilis as reference strains. The Viable Cell Staining Kit was validated with Escherichia coli, whereas the Total Cell Staining Kit was examined in selected experiments using both strains. Applicability to complex FMT matrices was confirmed using authentic fecal samples. Viable-cell counting showed strong linearity across a range of 9.4 × 106–1.2 × 108 cells/mL, with adequate accuracy and precision. Specificity was ensured by particle-size gating, unaffected by nonviable cells or diluent. Robustness was confirmed across operators and time points. Fecal samples showed linear quantifiability and acceptable precision; total-cell enumeration fulfilled linearity and precision requirements. The validated method enables rapid, reproducible viable-cell quantification suitable for Quality Control of FMT drug products, supporting formulation development, and dosing verification. Viable-cell quantification remains the analytical focus, despite limitations in spore detection. Total-cell enumeration provides complementary process information.
Keywords:
analytical method validation
fecal microbiota transplantation
GMP
ICH-Guideline Q2 (R2)
viable bacterial quantification
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