Expanded bed adsorption (EBA) is a preparative
chromatographic
In chemical analysis, chromatography is a laboratory technique for the separation of a mixture into its components. The mixture is dissolved in a fluid solvent (gas or liquid) called the ''mobile phase'', which carries it through a system ...
technique which makes processing of viscous and particulate liquids possible.
Principle
The
protein binding principles in EBA are the same as in classical
column chromatography and the common
ion-exchange
Ion exchange is a reversible interchange of one kind of ion present in an insoluble solid with another of like charge present in a solution surrounding the solid with the reaction being used especially for softening or making water demineralised, ...
,
hydrophobic interaction
In chemistry, hydrophobicity is the physical property of a molecule that is seemingly repelled from a mass of water (known as a hydrophobe). In contrast, hydrophiles are attracted to water.
Hydrophobic molecules tend to be nonpolar and ...
and
affinity chromatography
Affinity chromatography is a method of separating a biomolecule from a mixture, based on a highly specific macromolecular binding interaction between the biomolecule and another substance. The specific type of binding interaction depends on the ...
ligands can be used. After the adsorption step is complete, the fluidized bed is washed to flush out any remaining particulates.
Elution
In analytical and organic chemistry, elution is the process of extracting one material from another by washing with a solvent; as in washing of loaded ion-exchange resins to remove captured ions.
In a liquid chromatography experiment, for exa ...
of the adsorbed proteins was commonly performed with the
eluent flow in the reverse direction; that is, as a conventional packed bed, in order to recover the adsorbed
solutes
In chemistry, a solution is a special type of homogeneous mixture composed of two or more substances. In such a mixture, a solute is a substance dissolved in another substance, known as a solvent. If the attractive forces between the solvent ...
in a smaller volume of eluent. However, a new generation of EBA columns has been developed, which maintain the bed in the expanded state during this phase, producing high-purity, high yields of e.g. MAbs
onoclonal antibodiesin even smaller volumes of eluent. Process duration at manufacturing scale has also been cut considerably (under 7 hours in some cases).
EBA may be considered to combine both the "Removal of Insolubles" and the "Isolation" steps of the 4-step
downstream processing
Downstream processing refers to the recovery and the purification of biosynthetic products, particularly pharmaceuticals, from natural sources such as animal tissue, plant tissue or fermentation broth, including the recycling of salvageable com ...
heuristic. The major limitations associated with EBA technology is biomass interactions and aggregations onto adsorbent during processing.
Where classical column
chromatography
In chemical analysis, chromatography is a laboratory technique for the separation of a mixture into its components. The mixture is dissolved in a fluid solvent (gas or liquid) called the ''mobile phase'', which carries it through a system ...
uses a solid phase made by a packed bed, EBA uses particles in a
fluidized state, ideally expanded by a factor of 2. Expanded bed adsorption is, however, different from fluidised bed chromatography in essentially two ways: one, the EBA resin contains particles of varying size and density which results in a gradient of particle size when expanded; and two, when the bed is in its expanded state, local loops are formed. Particles such as whole cells or cell debris, which would clog a packed bed column, readily pass through a fluidized bed.
EBA can therefore be used on crude culture broths or slurries of broken cells, thereby bypassing initial clearing steps such as
centrifugation
Centrifugation is a mechanical process which involves the use of the centrifugal force to separate particles from a solution according to their size, shape, density, medium viscosity and rotor speed. The denser components of the mixture migrate ...
and
filtration
Filtration is a physical separation process that separates solid matter and fluid from a mixture using a ''filter medium'' that has a complex structure through which only the fluid can pass. Solid particles that cannot pass through the filter m ...
, which is mandatory when packed beds are used. In older EBA column designs, the feed flow rate is kept low enough that the solid packing remains stratified and does not fluidize completely. Hence EBA can be modelled as frontal
adsorption
Adsorption is the adhesion of atoms, ions or molecules from a gas, liquid or dissolved solid to a surface. This process creates a film of the ''adsorbate'' on the surface of the ''adsorbent''. This process differs from absorption, in which a ...
in a packed bed, rather than as a well-mixed, continuous-flow adsorber.
References
External links
"Expanded-bed adsorption" at Modern Drug Discovery
Introduction to Expanded Bed Adsorption
Biochemistry methods
Chromatography
Protein methods
{{chromatography-stub