Downstream Processing: Platform Technology for Developing Purification Processes - - BioPharm International

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Downstream Processing: Platform Technology for Developing Purification Processes


BioPharm International
Volume 20, Issue 3


Figure 6
Figure 6 illustrates the screening results with different salt conditions of a specific glycoprotein. The results show that most resins depict a similar binding profile—elution of the glycoprotein occurs around 100–200 mM NaCl. Based on these screening results, the best resins (with respect to binding capacity, throughput, cost price, etc.) are selected and studied in more detail in the next step, in which scouting experiments are carried out under dynamic conditions.

SCOUTING STUDIES

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 so on). 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).


Figure 7
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.


Figure 8A
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.


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