A High-Throughput Microbial Viability Assay to Evaluate Lyoprotectants for Live Biotherapeutic Products

Many cell-based therapies, such as live biotherapeutic products (LBPs), require preservation of cell viability during manufacturing, transportation, and storage before being delivered to patients. Although lyophilization is a common strategy to improve storage stability, it can compromise cell viability, thus reducing therapeutic efficacy. To mitigate this challenge, protective excipients, known as lyoprotectants, are necessary to protect cells during lyophilization. While current research centers on a few disaccharides such as trehalose and sucrose, there is a growing need to investigate and screen more alternatives to broaden the range of lyoprotectants and tailor formulations to specific strains. However, conventional methods for cell formulation screening, such as colony-forming assay, despite being considered as the gold standard for evaluating microorganism viability, are labor-intensive and expensive. In this study, we report a high-throughput screening (HTS) method based on well-plates to evaluate lyoprotectants for a representative LBP, BioPYM, a genetically modified Saccharomyces boulardii targeting gastrointestinal diseases. In this assay, various BioPYM formulations were lyophilized on a metal 96-well plate, washed to remove lyoprotectants, and then diluted and inoculated into a nutritional medium for growth monitoring. A significant inverse relationship was observed between log10(cell viability) and growth lag time, with a Pearson’s coefficient of −0.9862, suggesting that higher post-lyo viability can be reflected by shorter lag times during recovery growth. Comparable post-lyo viability was observed between well-plate-based lyophilization and bulk-material lyophilization. With this high-throughput method, we expect to boost the range of excipient choices beyond commonly used disaccharides to help cells withstand the stresses of lyophilization and more efficiently customize the protective formulations for cell-based therapeutics.