Experimental Design of the Multicolumn Countercurrent Solvent Gradient Purification (MCSGP) Unit for the Separation of PEGylated Proteins

The rapid growth and diffusion of biopharmaceuticals, including polyethylene glycol (PEG)-protein conjugates, required to set up stringent regulatory requirements, in terms of purity and clinical safety. In order to produce proteins that meet these requirements, the development of robust purification methods has been one of the main goals in the field of downstream processing in the last years. Most of these methods rely on single or sequential batch chromatographic separations, enabling one to reach the purity specification but often at the expense of low yield. An appealing alternative is the Multicolumn Countercurrent Solvent Gradient Purification (MCSGP). Thanks to the internal recycling of the fractions where product and impurities coelute, this process can provide a significant improvement in the process yield, preserving the purity specification. The drawback lies in the increased number of process parameters, compared to the batch, which complicates the design and optimization of this unit. In this work, we propose an ad hoc design procedure for the optimization of central-cut separations at a target purity. Using PEGylated lysozyme as a model system, we illustrate the use of this procedure to identify the load, the elution gradient as well as the collection intervals for optimal yield and productivity, at fixed purity specifications, for a batch (single) column system. The obtained optimal process parameters are subsequently transferred to the MCSGP unit. The so-designed MCSGP process exhibited superior performances compared to the corresponding optimal batch process by increasing the yield and productivity by 17.0% and 2.1%, respectively, at the purity specification of 80%.