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Materials Lecture 9 Separation

The document discusses downstream processing, which involves the recovery and purification of bioproducts following fermentation. Key operations include cell disruption, separation methods, and various filtration techniques to isolate and purify products. It also covers specific methods for cell disruption and the importance of selecting appropriate separation techniques based on the material state.

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6 views19 pages

Materials Lecture 9 Separation

The document discusses downstream processing, which involves the recovery and purification of bioproducts following fermentation. Key operations include cell disruption, separation methods, and various filtration techniques to isolate and purify products. It also covers specific methods for cell disruption and the importance of selecting appropriate separation techniques based on the material state.

Uploaded by

sixalid382
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© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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RECOVERY AND PURIFICATION OF PRODUCTS

Agnieszka Kuźniar
What is Downstream Processing ?

Downstream – ‘after the fermentation process’,primary ‘unit operations’ of Downstream Processing,

• Cell recovery/removal,

• Centrifugation,

• Dewatering,

• Ultrafiltration,

• Precipitation,

• Spray drying
What is Downstream Processing ?

The unit operations which are used to recover

bioproducts include those which facilitate

disintegration of solids, separation and recovery of

solids and liquids, recovery of soluble molecules

and the so-called finishing operations which include

processes such as drying and crystallization.


Typical downstream process for monoclonal antibody process
Cell Disruption

Disruption: the cell envelope is physically broken, releasing all intracellular components into the
surrounding medium
• There are several biotechnological products (vitamins, enzymes) which are located within the cells.
• Such compounds have to be first released (maximally and in an active form) for their further
processing and final isolation.
• The microorganisms or other cells can be disintegrated or disrupted by physical, chemical or
enzymatic methods.

The selection of a particular method depends on the


nature of the cells, since there is a wide variation in the
property of cell disruption or breakage.

For instance, Gram-negative bacteria and filamentous fungi can


be more easily broken compared to Gram-positive bacteria arid
yeasts.
Cell Disruption
Physical methods of cell disruption:

Impingement:
• In this procedure, a stream of suspended cells at high velocity and pressure are forced to
hit either a stationary surface or a second stream of suspended cells (impinge literally means
to strike or hit).
• The cells are disrupted by the forces created at the point of contact.
• Microfluidizer is a device developed based on the principle of impingement.
• It has been successfully used for breaking E. coli cells.
• The advantage with impingement technique is that it can be effectively used for disrupting
cells even at a low concentration

Microfluidizer
Cell Disruption
Physical methods of cell disruption:
Grinding with glass beads:

• The cells mixed with glass beads are subjected to a very high speed in a reaction vessel.
• The cells break as they are forced against the wall of the vessel by the beads.
• Several factors influence the cell breakage-size and quantity of the glass beads, concentration and age
of cells, temperature and agitator speed.
• Under optimal conditions, one can expect a maximal breakage of about 80% of the cells.
Material state and choice of separation methods

Material secreted ( ultrafiltration, centrifugation)

Not secreted material (cell disruption, solid-liquid separation)

Material in liquid ( ultrafiltration, adsorption)

Material in solid (extraction into aqueous solution)


Common Stages of Bioseparation
• Removal of solids
• Isolation (volume reduction)
• Purification
• Polishing
Typical Operations of Bioseparation
• Removal of solids – Filtration, centrifugation,
microfiltration
• Isolation of product (volume reduction) – Cell disruption,
extraction, adsorption,ultrafiltration, precipitation
• Purification – Adsorption, elution chromatography,
ultrafiltration, electrophoresis, precipitation, crystallization
• Polishing – Crystallization, drying, auxiliary process,
solvent recovery, water preparation
Biological
products Product
Nature of
bioseparation
required
Process filtration

• Filtration is used at several stages in the downstream processing of the bioreactor


harvest, as well as for the preparation of purified water and other processing fluids
(buffers, sanitizing agents, etc.).
• Several filtration steps are integral to the Capture, Intermediate purification, and
Polishing stages; these types of filtration fall into one of three general types:
Microfiltration can be used at the start of the downstream process to clarify the feed
beyond what was accomplished in the upstream harvest and centrifugation/clarification.
Ultrafiltration is used between chromatography steps to concentrate the product and
change the buffer conditions to prepare it for subsequent chromatography steps.
Sterilizing grade direct flow filtration, oftentimes involving the use of nanofiltration
cartridges, eliminates microbial organisms and insoluble proteins, removes adventitious
and endogenous viruses, and sterile filters the product in preparation for final
formulation
Flow-through devices, assembled with the above
membrane media, are formatted to affect two
general flow types:

Direct Flow Filtration devices allow the process fluid to cross the membrane in
essentially a perpendicular flow direction; this provides little or no prevention of
particulate build-up or the concentration of other elements that do not fit through
the pore structure.

Tangential Flow Filtration devices orient the membrane so that process flow
sweeps across the active filtration surface, which minimizes pore plugging and
surface fouling by concentrated reject elements of the feed.

Pall Life Sciences Ultipor® Direct Flow


Separation of soluble products

- Liquid-liquid extraction

- Precipitation

- Adsorption

- Membrane separation: ultrafiltration, dialysis, reverse osmosis

- Chromatography

- Electrophoresis

- Crystallization and drying


Dialysis
• Dialysis is a technique based on the diffusion of small
solutes from a concentrated solution to a lower-
concentration solution of this solute through a
semipermeable membrane until equilibrium is reached,
which is widely used in studies of in vitro drug release
studies.

• Delicate substances can be separated without damage


because dialysis is typically performed under mild
conditions: ambient temperature, no appreciable
transmembrane pressure drop, and low-shear flow.

• While slow compared with pressure-driven processes,


dialysis discriminates small molecules from large ones
reliably because the absence of a pressure gradient
across the membrane prevents convective flow through
defects in the membrane.
Dialysis

• An early industrial application of dialysis was caustic


soda recovery from rayon manufacturing.

• It had been a viable process because inexpensive but


alkali-resistant cellulose membranes were available
that were capable of removing polymeric impurities
from the caustic.

• Gradually however, dialysis is being replaced by


dynamic membrane technology for caustic soda
recovery because of the latter's much higher
productivity.
• Electrodialysis systems use a selectively permeable membrane to move ions from one side to

the other under the influence of an electric potential.

• In almost all practical electrodialysis processes, multiple electrodialysis cells are arranged into

a configuration called an electrodialysis stack, with alternating anion and cation exchange

membranes forming the multiple electrodialysis cells.


Thank you

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