From batch to continuous processing: purification of a bioactive peptide by means of Multicolumn Countercurrent Solvent Gradient Purification (MCSGP)

The interest around the use of peptides for therapeutic scopes is continuously increasing. Thanks to their high specificity and inherent affinity for target molecules, peptides represent an effective solution to unresolved medical issues avoiding typical toxic side effects of exogenous chemical drugs. From an industrial point of view, they are produced mainly by means of solid phase synthesis. However, during the synthesis, many undesired impurities (truncated or modified peptides, by-products, scavengers, etc.) are produced. Reversed-phase single-column preparative liquid chromatography is the preferred choice to obtain the target peptide at the desired degree of purity for pharmaceutical and therapeutic scopes. However, the presence of product-related impurities, chemically similar to the target, can generate several peak overlapping regions. In these cases, the purification is most likely governed by a yield-purity trade-off. This means that in order to obtain a pool with acceptable purity, the collection window need to be narrowed at the cost of yield (and vice-versa). In this work, we have investigated the possibility to overcome this trade-off limitation by applying twin-column Multicolumn Countercurrent Solvent Gradient Purification (MCSGP), an innovative method for purification based on the internal recycling of the overlapping regions between two columns connected in series. The presence of valves allows to alternatively operate the two columns interconnected or in batch mode (disconnected). The principle of operation is very simple: briefly, in the first column (upstream) the gradient is performed; the portion of the peak which respects the purity requirement is collected from the upstream column (in this moment the columns are disconnected); on the other hand the overlapping regions between product and weakly/strong adsorbed impurities (front and tail of the main peak) are recycled in the second downstream column which is also filled with fresh feed (in this phase the columns are interconnected). Then the two columns virtually change the position and another half-cycle starts by performing now the gradient in the second column. One full cycle is completed when the columns return in their original position (first column in upstream and second column in downstream). Steady-state conditions are usually reached after 4 or 5 cycles. These operations allow to increase recovery without losing purity. The design of the MCSGP methods is based on a single column batch chromatogram (named “design batch chromatogram”) where a portion of the main peak satisfies the purity requirements. This work will illustrate the optimization of experimental batch conditions for the purification in MCSGP of a raw glucagon mixture industrially synthesized. The results obtained with single columns batch purification will be compared to those obtained with MCSGP in terms of purity, yield and other important parameters such as solvent consumption. The effect of recycling and collection windows on the outcome of purification will be also discussed.