HPLC biogram analysis: a powerful tool used for hit confirmation in early drug discovery.

High-performance liquid chromatography (HPLC) biogram methodology is a powerful pharmaceutical screening hit confirmation strategy that couples analytical HPLC data with functional bioassay data. It is used primarily for screening hit chemical validation and triaging in support of early phase discovery programs and enables further investigation of the source of bioactivity in screening hits. The process combines semi-preparative separation technologies, automated compound handling and distribution, high-throughput biological screening, and informatics tools. The final output is an HPLC retention time versus bioactivity graphical overlay report. In this manner, biograms allow the analyst to determine which component in the sample is responsible for the biological activity, enabling decision making toward chemotype selection and prioritization from a pool of potential candidates. Another powerful aspect of the biogram assay lies in its utility in investigating biological activity in atypical samples, such as degraded samples or mixtures, for detection of minor active impurities or in addressing lot-to-lot activity discrepancies for a given test compound. Biograms are employed to track, isolate, and identify the source of biological activity in such samples, often yielding important information for program decisions.