Expanded Bed Adsorption Chromatography

Expanded Bed Adsorption (EBA) allows the protein removal directly from the fermentation broth without going for prior removal of particulates like cell debris and other unwanted products. It reduces the time and cost for further separation procedures in the down stream processing. We can eliminate the cell removal and clarification steps by adopting EBA. The scale-up of EBA is not so difficult, the adsorbent and equipment of EBA which resembles the conventional packed bed chromatography.
In biotechnological industries, the major steps which are following after upstream are removal of unwanted particulates and concentration of products. It is important to note down that the initial separation should be carried out to decrease the risk of proteolytic break down of the product. To achieve the particulates removal, centrifugation and/or micro-filtration are accepted as a good/standard in the biotechnology industry. But the long processing time and large unit requirement makes these methods not economical.

Flow diagram of EBA Chromatography
EBA

In EBA, the broth is applied as upward flow to make the settled adsorbent to expand which facilitates the more surface area of the absorbent and also making more space for cells and cell debris to pass through it without soaking the bed. The main advantage of the EBA over fluidized bed adsorption is back mixing. Many reaches are made to overcome this back mixing by making good column design and selection of adsorbent. The plug flow profile will be adopted to achieve the low back-mixing in the EBA column. Plug flow can be achieved by making pressure drop in the liquid inlet. Usually adsorbents for EBA are ranges from 50-400 microns. The adsorbent lists are given below, which is taken from the research paper of various journals. The column will be packed with larger adsorbent particles in the down and smaller in the top. Smaller adsorbent with more fluid velocities makes bed to expand high, larger adsorbents requires more liquid velocities to expand significant. Density of the adsorbent 1.1 to 1.3 g/ml is required to with stand at flow velocities and good productivity.

Properties of Commercial Adsorbants

To study the parameters of the EBA process, four different experiment methods will generally be adopted.
a. Small scale experiment in packed bed mode – to study the product characteristics on the purification process. Clarified broth should be used to avoid bed blocking. The optimal condition for binding and elution & binding capacity in presence of other proteins for the desired product will be studied by this step.
b. Small scale experiment in expanded bed mode – to find out the amount of biomass which can supply to the expanded bed and to access the ability of the process to remove cells and cell debris. To study the effect of DNA/debris/cells over the purification process.
c. Pilot scale experiment in expanded bed mode – to verify the results obtained from the small scale. ~25 – 200 liters of broth
d. Large scale experiment in expanded bed mode – to verify the pilot scale process results and performance identification. ~50-300 liters of broth.

The operation of EBA is similar to the packed bed column; the only difference is flow is upward for expansion, broth application and cleaning. In some research, they proved that by high velocity (Normally 300 cm/hr) the bed height is doubled to the packed height, which results in high productivity under some specific conditions. The elution is performed in the packed bed mode. After the unbound material has washed away, the bed will be allowed to settle down. The elution of proteins will be done from downward and velocity will be approximately 1/3rd of the expansion velocity, this facilitates the low consumption of buffers and cost effective. The upward elution can also be done; it will be 40% more buffer consumption than the previous.
Between every batch CIP has to be done, to avoid the carry over of materials between batches. CIP procedure depends on the absorbent substance and broth characteristics. Most CIP procedures include the usage of sodium hydroxide (0.5M to 1M) and a predicted application time. Sodium hydroxide normally used with other CIP solutions 10% acetic acid or 20% ethanol.
Here some applications of EBA over many products are given below (taken from some international journal) to understand better.

Common Applications of EBA

The monoclonal antibodies are capture by cation exchange chromatography or affinity chromatography on immobilized protein A. Thomess et.al reported the purpose designed matrix coupled with protein A for the recovery of monoclonal antibody through EBA. They studied 2mg of pure mouse IgG2 per cycle from an unclarified hybridoma cell culture using a 5 cm diameter expanded column. Only 150 ml of adsorbent was used to completely recover the antibody.

(Sincere thanks to guest editorASent for his contribution)

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