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.