Quantitative and Comparative Assessment of Recombinant Human β-Glucocerebrosidase Uptake Bioactivity Using a Stable...

Inconsistent conclusions on the cellular uptake of recombinant human β-glucocerebrosidase (rhGCase) for Gaucher disease stem from a fundamental limitation of existing methods: their inability to generate complete and reliable dose-response curves. This critical flaw, stemming from susceptibility to various experimental variables, prevents accurate potency comparison across different rhGCase products. To address this, we developed a robust bioassay using CHO-K1 cells stably expressing the human macrophage mannose receptor (hMMR). Our method quantifies uptake by measuring the enzymatic activity of internalized rhGCase and consistently produces a classic sigmoidal dose-response curve. Comprehensive validation and mechanistic studies, including inhibition experiments with mannose, fucose, and mannose-6-phosphate, confirmed that uptake is specifically mediated by hMMR, with successful enzyme transport to endosomes/lysosomes. Applying this assay to three commercial products yielded results contrary to prior literature: imiglucerase demonstrated superior uptake activity to velaglucerase alfa. The proposed method represents a significant improvement over existing assays, providing a more accurate and reproducible means to evaluate cellular uptake bioactivity, which is crucial for the quality control of rhGCase therapeutics.