The generally accepted method of purification of process streams for monoclonal antibodies includes capture of the product target with protein A, elution, and acidification to inactivate potential mammalian viruses, followed by cation-exchange chromatography and finally anion-exchange chromatography. There are some variations by manufacturers, which may include an intermediate hydrophobic interaction chromatography step.
As monoclonal antibodies are relatively similar from a structural perspective, purification processes tend to be somewhat consistent from manufacturer to manufacturer. However, with each processing step, the potential for heterogeneity is nonetheless introduced.
A recent report by the CMC Biotech Working Group, a consortium of companies that has prepared a case study on the development of a monoclonal antibody, outlined the steps involved in producing the monoclonal and the implementation of quality by design (QbD) concepts to optimize these steps and simultaneously control or eliminate process variation. So how can one identify and control these process variations—particularly structural variants—and simultaneously deliver a high-quality product with excellent yields?
Displacement chromatography (DC) is a method in which the components are resolved into consecutive rectangular zones of highly concentrated pure substances rather than solvent-separated peaks. The molecules are forced to migrate down the column by an advancing wave of a displacer molecule that has a higher affinity for the stationary phase than does the feed solute. Because of this forced migration, higher product concentrations and purities may be obtained compared to other modes of chromatography. There are distinct differences between displacement and elution chromatography.
DC has advantages over elution chromatography because the process takes advantage of the nonlinearity of the isotherms. A higher mass loading can be separated on a given column and the purified components recovered at significantly higher concentrations.
DC is no more complicated than elution chromatography. However, operating parameters are different, particularly in respect to optimization where displacer concentrations, flow rate, and protein load, are critical.
DC requires high loadings in order to set up a good displacement train. One typically uses loadings of 50% to 80% of the maximum resin capacity. If one does not have enough starting material to load at this level, then it is advisable to switch to a narrower column. Shorter columns will work in DC but recoveries will be lower.
Owing to the convergence of high loading, high recovery, and high purity of DC methods, one can obtain higher throughput per cycle, higher purity, and increased concentrations from each use of the column. This is useful if one needs to purify larger amounts of material and has the added advantage of achieving this with bench-scale analytical columns. However, the other advantage of DC is the ability to isolate trace components and concentrate them while simultaneously removing the main components.
Typically when producing antibodies with mammalian cell culture, structural and charge variants are encountered, which could potentially impact stability and activity of antibody. Many of these alterations are problematic to separate from the intended product and heroic methods are often utilized to obtain enough material to evaluate these discrepancies in pharmacodynamic studies. The thermodynamic properties of DC allow for enrichment of low concentration components and are applicable to isolating and purifying these charge variants.