Fluidized bed cchromatography involves the use of adsorbetn particles dispersed in a liquid medium. (Gagnon, J. Chromatogr. A 1221 (2012) 57-70).

Expanded bed chromatogrpahy (EBA): EBA is a type of stabilized fluidized bed chromatography. (Arpanaei, “improved expanded bed chromatography systems” thesis, 2007). EBA has found widespread appliations in the large scale purification of proteins from mammalian cell and microbial feedstocks in industrial bioprocessing. The technique is commonly employed in stirred-tank processes using particles with large particle diameters (0.5-mm) in order to separate adsorbent particles and biomass and to allow for a high capacity for a product. (Anspach, J. Chromatography A, 865 (1999) 129-144. Expanded bed chromatography differs from packed bed chromatoraphy in that  the upward flow of eluent provides that the adsorbent particles fall back down under gravity to form a stable bed whereas a downward flow of eluent in pack bed chromatography provides that the particles must be strong enough to support the entire column of adsorbent above them. Also, in expanded bed chromatography, the adsorbent is less crosslinked because it can be softer and more gel like which allows eluent to flow internally through the adsorbent as well as between the particles.

Expanded bed asorption reduces the number of operations in purificaiton processes by combining clarificaiton, concentration, and capture into one operation. It is based on stable fluidization and uses adsorbent particles with well-defined size and density distributions, otgether with columns designed to give even liquid flow distribution. The bed expands as the adsorbent particles are lifted by by an upward liquid flow through the column. The major benefit of using it is that adsorption can be carried out with unclarified feedstocks, there is no need for centrifugation or filtraiton to remove cells and debris. When the feedstock is applied, the target protein is captured by the adsorbent while cells and debris pass through the column unhindered. Washing is performed with the bed in an expanded mode, followed by elution of bound protein in a dedimented mode with downward flow. The choice of ligand is not limited to ion-exchangers. It is possible to sue hydrophobic, chelating and affintiy ligands. However, the feedstocks sued with expanded bed are usually much cruder than the feedstocks of traditional packed bed chromatography; the ligands must be abel to witstand a vigrous cleaning procedure. Frej (Biotech & Bioengineering 44 (1994) 922-929)

Chromatography Schemes Using EBA

Protein A EBA: There is significant increased in the dynamic binding capacity when Protein A EBA is operated at slower flow rates. Blank (Bioseparation 10: 65-71, 2001)

Breakthrough studies on streamine Protein a using purified antibody in packed-bed mode and unclarified cell culture fluid in the EBA showed very similar DBCs. Blank (Bioseparation 10: 65-71, 2001)

Fahrner (J. Biotechnology 75 (1999) 273-280) discloses that wehreas a packed bed protein A affinity column must have cells and cell debris removed from the cell culture fluid prior to loading, often by tangential flow filtration, the abiliyt to load unclarified cell culture fluid onto the protein A affinity column can eliminate a unit operation and thus could improve production efficiency and reduce production cost. Fahrner discloses that EBA protein A using Streamline rProtein A media is an efficient method for prufiying mAb from CHOPs comparible to packed bed. The DBC of the EBA is related to flow rate (measrued in column volues per hour) by a power function, which allows a high capacity at a low flw rate. At 250 cmh-1 with a 25 cm bed hight, teh DBC is 30 g/l

A 3 step process with Protein A EBA results in the same purity as the conventional prcoess. Blank (Bioseparation 10: 65-71, 2001)

–Protein A(EBA) -CEX – AEX: 

–Protein A EBA – CEX – AEX: 

CEX EBA: 

A pH of 4.5 and conductivity of 10 mS are reasonable parameters for manufacturing operations and given the accpetable DBC of 23 mg/ml. Blank (Bioseparation 10: 65-71, 2001)

Effect pH: At a pH to 4.75 at 10 mS improved the DBC to 15 mg/ml. At pH 4.5 the DBC was greater than 20 mg/ml. Blank (Bioseparation 10: 65-71, 2001)

Effect of conductivity: The DBC may also be increased by decreasing the conductivity. Blank (Bioseparation 10: 65-71, 2001)

–CEX EBA -Protein A – AEX: Blank (Bioseparation 10: 65-71, 2001) disclose that a 3 step process with CEX EBA as the capture step cannot remove antibody aggregate and leached Protein A to acceptable levels.