EBA Chromatography Systems (Website Content)
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EBA Chromatography Systems Combines Three Stages Into One, Saving Time and Reducing Costs of Purification
Expanded-bed adsorption (EBA) chromatography systems are proving to be valuable, time- and money-saving capital investments for companies in the business of purifying proteins and peptides. EBA technology not only improves process economy, but produces better results and higher yields by clarifying, concentrating and purifying feedstock in one step.
Every step in the purification process adds additional expense, time and human resources. Generally, viscous feedstocks are clarified before a chromatography step by combining centrifugation and filtration operations. This multi-step process increases process times and results in some product loss.
This keeps the scale of equipment small and reduces cycle time. EBA provides a robust and highly efficient, single-step procedure for extraction and purification of recombinant proteins from unclarified particulate-containing feedstocks, removing expensive preclarification, concentration, and purification steps.
Expanded bed adsorption was originally conceived as a first capture step compatible with crude bio-feedstock containing a mixture of insoluble and soluble biological molecules such as proteins, genomic DNA fragments, RNA molecules, and cell debris.
How EBA Works
First, adsorbent particles are fluidized to provide a density distribution of a defined size, referred to as an expanded bed. An upward liquid flow expands adsorbents at a velocity corresponding to its sedimentation velocity forming a stable fluidized bed. The degree to which a bed expands depends on size and density of adsorbent beads as well as the linear flow velocity and viscosity in the mobile phase.
A bed is considered stable when only small movements of adsorbent beads are observed.
Unclarified feed is applied to the expanded bed with the same upward flow as used during expansion and equilibration. The ultimate system performance, mixing process, and bed stability is largely dependent on the introduced quality of flow within the column. Processing time is also critical at this stage. Fermentation broths and crude cell homogenates contain proteases and glycosidases that can reduce product recovery. This also creates degradation products that may be difficult to remove later.
Adsorbents binds target proteins while particulates, contaminants, and cell debris pass through unhindered. The upward liquid flow allows them to be washed out from the expanded bed.
Liquid flow is then stopped allowing adsorbent particles to quickly settle within the column.
Flow is then reversed. Using suitable buffer conditions captured proteins are eluted from the sedimented bed, clarified, partly purified, and ready for further purification via packed-bed chromatography.
After elution, downward flow, using buffers specific for the type of chromatographic principle applied, washes and regenerates the bed.
Finally, in order to remove nonspecifically bound, precipitated, or denatured substances from the bed, and restore it to its original performance, a clean-in-place procedure is applied, leaving the bed refreshed.