Chromatography is the most important unit operation in the downstream processing of biopharmaceuticals. In most cases, at least two chromatography steps are required to complete the purification: a so-called capture step and a polishing step. The purpose of the capture step is the concentration of the product and the removal of the largest part of the non-product-related impurities. The purpose of the polishing step is the removal of the remaining impurities, in particular of product-related impurities such as aggregates and fragments.
Traditionally, the capture and polishing chromatography steps are carried out in batch mode, having inherent drawbacks in achievable capacity, yield, and purity. Countercurrent chromatography is an ultra-high resolution chromatographic principle, which even when operated with only two columns can overcome these drawbacks to a large extent.
For capture steps, affinity chromatography is the method of choice. Since affinity chromatography is highly specific, it can purify the desired product with high yield. However, due to the elaborate and chemically susceptible ligands, affinity chromatography stationary phases are rather expensive. Protein A affinity chromatography is an exception as its capacity and chemical stability has been steadily optimized over the past decades, and commercial competition has lowered stationary phase prices.
Other affinity chromatography materials for newer applications, such as for the capture of mAb fragments, have not yet evolved this far and have significantly lower capacities and lower chemical resistance while being expensive.
Therefore, in particular for capture processes, it is of great importance to maximize stationary-phase capacity utilization in order to save cost and reduce processing time.
The dynamic binding capacity (DBC) is strongly dependent on the residence time of the product in the column. An increased feed flow rate increases the throughput of the process but decreases the residence time and thereby the breakthrough point, representing the point whereupon product is lost at continued loading. The breakthrough point is typically expressed as 1% DBC, which corresponds to the binding capacity achieved when the column is loaded until the breakthrough concentration exceeds 1% of the feed concentration.
In batch downstream processing, resin capacity is not used optimally as feed volumes of 80–90% of the 1% DBC value are loaded in order to avoid breakthrough.