Residence time distribution in counter-current protein A affinity chromatography using an inert tracer.

The trend in the biopharmaceutical industry is changing from batch process to continuous process. For continuous biomanufacturing, traceability of the material is required by regulatory authorities. The recent ICH draft guideline Q13 on continuous manufacturing of drug substances and drug products requests an "understanding of process dynamics as a function of input material attributes (e.g., potency, material flow properties), process conditions (e.g., mass flow rates) … One common approach is characterization of residence time distribution (RTD) for the individual unit operations and integrated system." Thus, it is necessary to trace material through individual continuous unit operations and the integrated process. The RTD of a process is obtained experimentally by injecting a pulse of an inert tracer into the inlet and measuring the broadening of the injected pulse in the outlet. We investigated the RTD of three-column periodic counter-current chromatography (PCC) using staphylococcal protein A affinity chromatography, with a focus on how the material distributes over subsequent cycles. A fluorescent-labeled antibody was used as the inert tracer under high salt concentration. The tracer was injected once in each run but at different points of the loading phase. We then analyzed the outlet of the column. In the elution phase, regardless of the point of injection, we observed an even distribution of the tracer. In the loading phase, a constant exchange between the antibody in the solid phase and the liquid phase was observed, meaning that sending the outlet of one chromatography column into another column to improve resin utilization causes higher residence time in the system for some portion of the material.