Increasing upstream yields have led to the need for downstream purification capacities. In this article, a new chromatographic process for the purification of biomolecules is presented. Its working principle is explained and possible areas of application in monoclonal antibody and polypeptide purification are discussed. For an industrial polypeptide purification problem, experimental work showed that a multicolumn countercurrent solvent gradient purification (MCSGP) process could raise the productivity 25-fold, compared to the current batch chromatographic step in production.
The majority of the therapeutic proteins undergoing late-stage development or entering the market are monoclonal antibodies (MAbs). Because high doses of these products are needed per patient, relatively large amounts of protein must be produced and purified. The past decade saw tremendous advances in cell culture technology producing higher yields. As a result, purification has now become a bottleneck, or at least a major cost driver with a high potential for cost cutting. Consequently, MAb purification has received a lot of attention in academia and industry and a variety of solutions have been proposed, including but not limited to crystallization, (i.e., ion-exchange), Protein-A mimetic ligands, liquid–liquid extraction, and non-affinity chromatography in batch or continuous operation. The non-affinity chromatography used in a continuous process such as multicolumn countercurrent solvent gradient purification (MCSGP) shows high potential to replace current standard MAb purification schemes, as seen in a recent presentation by Merck-Serono.1–8
Conventionally, the purification of polypeptides is performed using reversed-phase chromatography in a batch operation. Often, two or more chromatographic steps with different mobile phases are used to achieve the specified purity. Continuous processes are ideally suited for such difficult purification steps because they can significantly improve the separation efficiency compared to conventional batch chromatography. The best known continuous chromatographic process is the simulated-moving-bed (SMB) process.10 In the pharmaceutical industry, that method has been applied successfully at commercial scale for the purification of small, chiral molecules. The challenge in such purifications is to separate two enantiomers from a racemic mixture. But the SMB technology is generally of no use in the purification of therapeutic polypeptides or proteins because it can perform only binary separations where gradient chromatography cannot be properly implemented. For very specific purification problems, however, academia has investigated the use of SMB and SMB-derivatives for the purification of biomolecules.11–15 A detailed comparison of the existing continuous chromatographic process has been published elsewhere.6
It is also worth noting that the SMB technology can only perform binary separations i.e., producing two streams, such as an early stream eluting waste and a later stream/eluent product or two product streams (product A and product B). The purification of therapeutic polypeptides and proteins in non-affinity chromatography, however, requires ternary separations, i.e., three streams containing the early eluting waste, the product, and the late eluting waste. Unlike the SMB process, the MCSGP process is suited for ternary separations using gradient chromatography and can therefore be used for the continuous purification of therapeutic polypeptides and proteins.
Recently, the MCSGP process was tested for the purification of the polypeptide Calcitonin (a growth hormone) in a joint project with Novartis Pharma AG (Basel, Switzerland), ChromaCon AG and ETH Zurich (both in Zurich, Switzerland).
In this article, the purification challenges for the reversed-phase chromatography of Calcitonin are described and the experimental results of the purification of this polypeptide with the MCSGP process are shown. A comparison is given in terms of purity, yield, and productivity between the MCSGP process and the conventional batch process for the purification problem presented.