The presentation deals with one of the latest techniques called expanded bed chromatography.The need, mechanism, advantages and applications of the technique is very well described.

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2. *The majority of biotechnology processes for producing pharmaceutical or
diagnostic product involve the purification of proteins and peptides .
*The sources includes bacteria, yeast and mammalian cell culture fluids, or
extracts from naturally occurring tissue.
**The initial purification of the target molecule necessitates clarification of the crude
sample before application to the chromatography.
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3. The standard techniques used for removal of cells and/or cell debris have been
centrifugation and microfiltration.

4. The standard techniques used for removal of cells and/or cell debris have been
centrifugation and microfiltration.
Problem?

5. The standard techniques used for removal of cells and/or cell debris have been
centrifugation and microfiltration.
Problem?
*small particle size and high
viscosity reduce the efficiency
*Difficult to obtain a completely
particle-free liquid.
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6. The standard techniques used for removal of cells and/or cell debris have been
centrifugation and microfiltration.
Problem?
**The flux of liquid per unit
membrane area is often
decreased in later periods and
hence is time
consuming.
**Fouling of the microfiltration
membranes significantly adds
to the operational cost.
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7. The standard techniques used for removal of cells and/or cell debris have been
centrifugation and microfiltration.
Solution?
Combined use of centrifugation and filtration

8. The standard techniques used for removal of cells and/or cell debris have been
centrifugation and microfiltration.
Solution?
Combined use of centrifugation and filtration
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9. The standard techniques used for removal of cells and/or cell debris have been
centrifugation and microfiltration.
Solution?
Combined use of centrifugation and filtration
*Long process times
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10. The standard techniques used for removal of cells and/or cell debris have been
centrifugation and microfiltration.
Solution?
Combined use of centrifugation and filtration
*Long process times
*Large units causing significant
capital expenditure
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11. The standard techniques used for removal of cells and/or cell debris have been
centrifugation and microfiltration.
Solution?
Combined use of centrifugation and filtration
*Long process times
*Large units causing significant
capital expenditure
*Results in significant product loss
due to product degradation.
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15. (1) The beads are embedded and are stationary.
http://pubs.acs.org

16. (1) The beads are embedded and are stationary.
(2)As the column is fluidized, the resin beads establish a
concentration gradient
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17. (3)The sample feedlot is injected, and particulates and cell debris
(green dots) move past the resin and out of the column, while the
compound of interest (red dots) interacts with the beads
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18. (3)The sample feedlot is injected, and particulates and cell debris
(green dots) move past the resin and out of the column, while the
compound of interest (red dots) interacts with the beads
(4)The column is then repacked, the flow is reversed, and the
compound is eluted from the beads
http://pubs.acs.org

19. * Allows the protein removal directly from the fermentation broth without
going for prior removal of particulates like cell debris and other unwanted
products.
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.

20. * Allows the protein removal directly from the fermentation broth without
going for prior removal of particulates like cell debris and other unwanted
products.
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.
It reduces the time and cost for further separation
procedures in the down stream processing.

21. * Allows the protein removal directly from the fermentation broth without
going for prior removal of particulates like cell debris and other unwanted
products.
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.
It reduces the time and cost for further separation
procedures in the down stream processing.
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22. **The resin is confined between the
bottom of the column and the flow
adapter, clogging occurs when particulate
matter and cell debris cannot flow around
the closely packed resin beads.
**EBA columns are fed from below, and the
adapter is held away from the packed resin
level, giving the resin room to expand and thus
creating spaces between the beads.
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23. (1) EBA was also useful for the purification of large quantities of plasmid
DNA from bacteria.
Steps :
*Lyse the bacteria with a detergent solution.

24. (1) EBA was also useful for the purification of large quantities of plasmid
DNA from bacteria.
Steps :
*Lyse the bacteria with a detergent solution.

25. (1) EBA was also useful for the purification of large quantities of plasmid
DNA from bacteria.
Steps :
*Lyse the bacteria with a detergent solution.
* Run the solution over an anion exchange EBA
column.

26. (1) EBA was also useful for the purification of large quantities of plasmid
DNA from bacteria.
Steps :
*Lyse the bacteria with a detergent solution.
* Run the solution over an anion exchange EBA
column.
Result : This yielded a plasmid DNA solution that was one step away from being suitable for
human clinical trials without the use of typical toxic reagents such as phenol, ethidium bromide
and cesium chloride.

27. (2) Application in diagnostics and epidemiology
*The researchers used affinity EBA and created a
monoclonal antibody that bound a protein on the
surface of the spores of Enterocytozoon bieneusi, an
opportunistic protistan parasite that causes
diarrhea, malabsorption, and weight loss.
*Conjugating this mAb to Protein adsorbent, French
group researchers isolated spores from numerous
human stool samples, detecting the spores by an
indirect immunofluorescence antibody test.
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28. *Several companies are looking forward to create variations on the ion-exchange
resins that will allow more specific binding of products and limit
the effects of salt concentration.
*Increasing the volumes that can be run through the system is another
issue to be worked upon.
*Making EBA more suitable for the loading of samples directly from
fermenters can also aid expanding the applications.
“Beyond this, the future seems to lie in
an expanded line of affinity chromatography matrices’.

29. ** “Expanded-bed chromatography in primary protein purification”
By F.Birger Anspach, , David Curbelo, Ralf Hartmann, Gunnar Garke, Wolf-Dieter
Deckwer
**“Physicochemical Basis of Expanded-Bed Adsorption for Protein
Purification” Handbook of Bioseparations, Vol. 2: Separation Science and
Technology; S. Ahuja, Ed.; Academic Press: San Diego, 2000.
**“Expanded-Bed Adsorption Process for Protein Capture” Handbook of
Bioseparations, Vol. 2: Separation Science and Technology; S. Ahuja, Ed.;
Academic Press: San Diego, 2000.
**“Expanded-bed adsorption” article by Randall Willis
**http://www.desmech.com/
libguides.library.curtin.edu.au

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