The reference output (\(y_{\text{ref}}\)) was also determined online during the experiment after process startup. The previously calculated PCC cycle time was applied during the first few cycles until steady state had been reached. The area of the eluate peak from the polishing step was then automatically calculated and was used to calculate the product output, which was set as the reference signal.

Pooling control

The cutoff times of the pooling of the product in the polishing step were obtained based on the UV absorbance at a wavelength of 280 nm, measured online with a U9-M UV monitor, i.e., pooling started and ended at a certain value of absorbance, as described previously (Löfgren et al., 2018). In addition, in the present study, the second absorbance cutoff (i.e., when pooling ends) was automatically adjusted to compensate for the variation in the incoming concentration. The height of the eluate peak was obtained online, and the absorbance cutoff was calculated based on the height. Previous runs of the process were used to determine the linear relationship between the absorbance cutoff and the peak height.