Examining pre-analytical factors for bioanalysis of protein therapeutics crossing the blood–brain barrier through receptor-mediated transcytosis

BACKGROUND: The blood-brain barrier (BBB) poses challenges for delivering large-molecule therapeutics to the brain. While engineered cross-BBB delivering platforms such as antibodies utilizing receptor-mediated transcytosis (RMT) aim to improve drug delivery, accurate quantization of antibody exposure in the brain is critical. This study investigates pre-analytical factors impacting the analysis of BBB-shuttling antibodies in mice and seeks to bring attention to steps that can be optimized to enhance efficiency and throughput. RESEARCH DESIGN AND METHODS: We evaluated factors such as perfusion, capillary depletion, and freeze-thaw in a humanized mouse model treated with antibodies, with or without a BBB shuttle. Hemoglobin and albumin were investigated as markers for blood contamination. RESULTS: Blood contamination was effectively reduced when cardiac puncture was performed, and perfusion further minimized the contamination. Albumin is a more representative blood contamination marker than hemoglobin for antibody bioanalysis in brain tissue. Capillary depletion did not significantly affect PK analysis under-tested conditions, and no considerable differences were found between frozen and fresh samples. CONCLUSIONS: Careful investigation of these pre-analytical procedures should be conducted to understand their impact. This will facilitate the development of an optimized and efficient workflow for discovery screening.