Downstream Processing_basic introduction.ppt

Downstream Processing_basic introduction

1. Downstream Processing
The manufacture of human proteins by the methods of
modern biotechnology is separated into two stages:
1.upstream processing during which proteins are
produced by cells genetically engineered to contain
the human gene which will express the protein of
interest (Molecular BT)
2.downstream processing during which the produced
proteins are isolated and purified (Industrial BT)

2. Fig: A generalized block diagram of downstream processing

3. SYNTHESIS OF BIOSEPARATION
PROCESSES
Experienced engineers heavily rely on certain rules of thumb,
also known as heuristics, for putting together the skeleton of a
recovery and purification process. A few such heuristics follow:
1. Remove the most plentiful impurities first.
2. Remove the easiest-to-remove impurities first.
3. Make the most difficult and expensive separations last.
4. Select processes that make use of the greatest differences
in the properties of the product and its impurities.
5. Select and sequence processes that exploit different
separation driving forces.

4. CELL HARVESTING
* Centrifugation
* Microfiltration
* Ultrafiltration
CELL DISRUPTION
* Homogenization
* Bead Milling
* Osmotic Shock
CELL DEBRIS REMOVAL
* Centrifugation
* Microfiltration
* Vacuum Filtration
* Press Filtration
BIOMASS REMOVAL
* Vacuum Filtration
* Centrifugation
* Microfiltration
* Ultrafiltration
* Press Filtration
* Candle Filtration
* Flotation
PRODUCT EXTRACTION BY
* Aqueous Two-Phases
* Organic Solvents
* Expanded Bed Adsorption
* Batch Adsorption
* Supercritical Fluids
* Reverse Micelles
* Extractive Distillation
CONCENTRATION
* Ultrafiltration
* Evaporation
* Reverse Osmosis
* Precipitation
* Crystallization
* Extraction
* Adsorption
* Distillation
RENATURATION
* Dissolution
* Refolding
FINAL PURIFICATION
* Chromatography (Affinity,
Reversed Phase, Ion Exchange,
Size Exclusion, etc.)
* Diafiltration
* Electrodialysis
* Electrophoresis
DEHYDRATION OR
SOLVENT REMOVAL
* Spray Drying
* Freeze Drying
* Tray Drying
* Fluid Bed Drying
* Drum Drying
BIOREACTOR
Intracellular Extracellular
Products Products
High Purity Low Purity
Required Required
IB’s
For Solid
Final Form
Fig: A generalized block diagram of downstream processing

5. Fig: Folded and denatured protein

6. Downstream Processing
Downstream processing is any treatment of culture broth after
fermentation to concentrate and purify products. It follows a
general sequence of steps:
1. Cell removal: (filtration, centrifugation)
2. Primary isolation: to remove components with properties
significantly different from those of the products (adsorption,
liquid extraction, precipitation). Large volume, relatively non
selective
3. Purification: Highly selective (chromatography, ultra filtration,
fractional precipitation)
4. Final isolation: (crystallization, followed by centrifugation or
filtration and drying). Typical for high-quality products such as
pharmaceuticals.
Downstream processing mostly contributes 40-90 % of total cost.

7. What is Downstream Processing?
•Downstream – ‘after the fermentation process’
•Primary ‘unit operations’ of Downstream
Processing
 Cell recovery/removal
Centrifugation
 Dewatering
Ultrafiltration
Precipitation
Spray drying

8. Downstream Processing
•Secondary ‘unit operations’
 Protein purification
Adsorption chromatography
Gel permeation chromatography
 Protein processing
 Immobilisation
 Beading/Prilling
 Protein packaging
Sterilisation
 Bottling etc

9. Separation of cells and medium
•Recovery of cells and/or medium (clarification)
For intracellular enzyme, the cell fraction is required
For extracellular enzymes, the culture medium is
required
•On an industrial scale, cell/medium separation is
almost always performed by centrifugation
 Industrial scale centrifuges may be batch, continuous,
or continuous with desludging

10. Industrial centrifuges
Tubular bowl Chamber Disc

11. Properties of industrial centrifuges
•Tube
High centrifugal force
Good dewatering
Easy to clean
•Chamber
Large solids capacity
Good dewatering
Bowl cooling possible
•Disc type
Solids discharge
No foaming
Bowl cooling possible
Limited solids capacity
Foams
Difficult to recover protein
No solids discharge
Cleaning difficult
Solids recovery difficult
Poor dewatering
Difficult to clean

12. Centrifugation properties of different cell
types
•Bacteria
Small cell size
Resilient
•Yeast cells
Large cells
Resilient
•Filamentous fungi
Mycelial
Resilient
•Cultured animal cells
Large cells
Very fragile
High speed required
Low cell damage
Lower speed required
Low cell damage
Lower speed required
High water retention in pellet
Very susceptible to damage

13. Dewatering
•Dewatering of whole cell fraction (use centrifugation)
•Dewatering of culture medium or a lysed cell fraction (for
recovery of a soluble protein fraction)
 Precipitation
Salting out – addition of a high concentration of a soluble salt
(typically ammonium sulphate) causes proteins to aggregate
and precipitate
 Ultrafiltration
The solution is forced under pressure through a membrane
with micropores, which allows water, salts and small
molecules to pass but retains large molecules (e.g., proteins)
 Spray drying
Requires use of heat to evaporate water – unsuitable for most
proteins

14. Cell disruption (for intracellular enzymes)
•Sonication
Use of high frequency sound waves to disrupt cell walls and
membranes
Can be used as continuous lysis method
Better suited to small (lab-scale) operations
Can damage sensitive proteins
•Pressure cells
 Apply apply high pressure to cells; cells fracture as pressure is
abruptly released
Readily adapted to large-scale and continuous operations
Industry standard (Manton-Gaulin cell disruptor)
•Enzymic lysis
 Certain enzymes lyse cell walls
Lysozyme for bacteria; chitinase for fungi
Only useful on small laboratory scale

15. Protein purification
•Adsorption chromatography
Ion exchange chromatography – binding and separation
of proteins based on charge-charge interactions
 Proteins bind at low ionic strength, and are eluted at
high ionic strength
+
+
+
+
+
+
+
+
+
+
-
-
-
-
+
+
+
+
+
+
+
+
+
+
-
-
-
+
Positively charged
(anionic) ion
exchange matrix
Net negatively
charged (cationic)
protein at selected pH
Protein binds to matrix

16. Reminder about protein net charge, pI and pH
•All proteins have ionisable groups on the surface (N-
terminal amino and carboxylate, Glu, Asp, His, Lys and
Arg side chains)
•These groups are charged or neutral depending on pH
(e.g., -COO
-
+ H
+
 COOH)
•The net charge on a protein changes at different pHs
•Each protein has a pH where the net charge is zero (the pI:
Isoelectric Point)
•Useful rules:
 At pH > pI, protein net charge is negative
 At pH < pI, protein net charge is positive
 At pH = pI, protein net charge is zero

17. Typical ion exchange protein separation
Loading starts
Loading ends,
Low salt wash begins
Protein absorbance
Peak of
unbound
protein
Salt gradient
0
1M
Salt gradient
begins
Salt gradient
ends
Eluted peaks of weakly bound (I),
moderately bound (II)
and tightly bound (III) proteins
II
III
I

18. Affinity chromatography
•Binding of a protein to a matrix via a protein-
specific ligand
 Substrate or product analogue
 Antibody
 Inhibitor analogue
 Cofactor/coenzyme
•Specific protein is eluted by adding reagent which
competes with binding

19. Affinity chromatography
Matrix Spacer arm
Affinity
ligand
+
Active-site-bound enzyme
1. Substrate analogue affinity chromatography
Matrix Spacer arm
Antibody
ligand
+
Antibody-bound enzyme
2. Immunoaffinity chromatography
Protein epitope
Enzyme

20. Gel permeation chromatography (GPC)
•Also known as ‘size exclusion chromatography’
and ‘gel filtration chromatography’
•Separates molecules on the basis of molecular size
•Separation is based on the use of a porous matrix.
Small molecules penetrate into the matrix more,
and their path length of elution is longer.
•Large molecules appear first, smaller molecules
later

21. GPC in operation
Large protein Small protein
Short path length Longer path length

22. Downstream processing depends on
product use
1.Enzyme preparations for animal feed
supplementation (e.g., phytase) are not purified
2.Enzymes for industrial use may be partially
purified (e.g., amylase for starch industry)
3.Enzymes for analytical use (e.g., glucose
oxidase) and pharmaceutical proteins (e.g., TPA)
are very highly purified

23. Fermentation
Culture supernatant
Centrifugation
to remove cells
Liquid preparation
to animal feed
market
Fermentation
Culture supernatant
Fermentation
Cell pellet
Intracellular fraction
Animal feed enzyme Analytical enzyme Therapeutic protein
Centrifugation
to remove cells
Centrifugation
to remove
medium
Protein
precipitation
Cell
lysis Centrifugation
Protein fraction
Protein
precipitation
Protein fraction
1 or 2 purification
steps
Semi-purified
protein 3-4 purification
steps
Homogeneous
protein
Sterile
bottling
To pharmaceuticals market
Lyophilisation
Bottling
To chemicals market

24. Operational diagram of large-scale fungal
batch fermentation system
Preculture Preparation of Fermentation Recovery of enzyme-
inoculum containing medium