In the scouting experiments, the most suitable resins are studied in greater detail in packed bed columns. The technique became
available in the second part of the nineties following the commercialization of new purification systems such as the Akta
design (GE Healthcare, Uppsala, Sweden).
Scouting studies are carried out by running the columns in a series with different buffer conditions (salt, pH, buffer, and
soon). During both the screening and scouting stages, it is also possible to fine-tune the selected ligand of the resin (e.g.,
selecting hydrophobic or ionic ligands). The purification results from a hydrophobic interaction step (i.e., scouting various
hydrophobic ligands) for a specific glycoprotein are shown in Figure 7.
As observed from the elution profiles, the target protein elutes in the salt gradient depending on the structure of the ligand
coupled to the resin. With some ligands, the target protein elutes partly in the regeneration phase, together with other proteins.
Most host cell proteins remain bound to the column and elute mainly after finishing the salt gradient. After these scouting
studies, the best chromatographic resin is selected and further optimization occurs with experimental design approaches.
Statistical Design Studies
Experimental design is a statistical approach that uses various matrices; response surface and screening designs are the most
preferred methods.8 Screening designs are normally used to analyze large numbers of parameters to determine which are the most critical; at
first, only the main effects of the parameters are tested. The selected parameters are then analyzed in more detail with a
response surface design that handles fewer parameters (the most critical ones), but also determines the influence of the interactions
between parameters. Overall, experimental design is needed to increase the robustness of purification processes.
In the example below, the most important parameters—such as pH, the salt and protein concentrations in the loading phase,
and the pH and salt in the elution phase—of a specific purification step for a protein, are analyzed with a response surface
design. Figure 8 shows the effects of the main parameters (pH,conductivity, protein) in the loading and elution phases on
product content. The results in Figure 8A clearly show the effects of the main parameters—pH,conductivity, salt—in the loading
(elution) phase over a broad range around the standard operating ranges. The outcome of these studies can be used to set or
restrain limits for certain critical parameters that may influence or become critical in this specific purification step.
Moreover, optimization in terms of interaction effects, as shown in Figure 8B (interaction between pH in loading phase and
conductivity in the elution phase) indicate that not only are the main parameters important, but their interactions are, too.
Finally, this platform technology shows that a structural approach for setting up a downstream process (for each purification
step) is feasible. Speed, understanding, and robustness may aid in the use of this platform technology as a very useful approach
for every downstream process in development.
This article first appeared in BioPharm Int. 2007 Mar;20(3)44-50.
MICHEL H.M. EPPINK is director of the downstream processing methodology and troubleshooting section of the API/biotech division at NV Organon,
Oss, The Netherlands,+31 412 665850, email@example.com
RICK SCHREURS is group leader of the section, and ANKE GIJSEN and KEES VERHOEVEN are research technicians.