The biotechnology community has recently gained tremendous interest in Process Analytical Technology (PAT) due to the resulting increased consistency in product quality from run-to-run. Since protein products are usually expressed in a non-native organism, the synthesis and purification of them require careful monitoring of product quality at each stage of the process. This talk focuses on a case study where an on-line HPLC system was tested for its feasibility for performing at-line analysis and subsequent pooling for a hydroxyapatite chromatography purification step. The results obtained using the on-line HPLC were successfully verified by performing analysis via an off-line HPLC. The results showed that it is feasible to use the on-line HPLC for the application under consideration in the manufacturing environment.