BACKGROUND: Recently, there has been increased interest in employing process analytical technologies (PAT) that combine an understanding of how operating parameters affect product quality together with real-time analysis to achieve efficient process manipulation during operation and thereby ensure that product outputs meet specifications. Primarily intended as the basis of control strategies for full-scale manufacturing systems, such techniques can bring significant benefits for steps such as chromatographic separations. Rapid at-line monitoring of a target product can also enable the design of more sophisticated development trials than are generally used at present, whereby data gathered during development can be used to initiate the use of control techniques that drive a process to achieve target values for outputs such as yield or purity. Furthermore, the acquisition of rapid at-line data also frees the operator from having to perform potentially laborious off-line assays. RESULTS: To demonstrate the value of such an approach, a target product (alcohol dehydrogenase) produced in Saccharomyces cerevisiae and purified by hydrophobic interaction chromatography (HIC) was monitored in real time by stopped-flow analysis (SFA) every 45 s. The at-line monitoring system of the HIC step used in both packed and expanded bed modes provided a visual description of product levels, facilitating a real-time representation of yield. CONCLUSION: This PAT information enabled process control wherein the load cycle was regulated successfully to a pre-specified set-point to achieve a desired level of breakthrough and so ensure that the product met its target specification. (c) 2009 Society of Chemical Industry