Downstream Processes
BIE 5900/6600
Spring 2010
Products
• From plant, animal or microbial cells
• Biomass
– Algae for anaerobic digestion and for biodiesel
– Anaerobic digester sludge for land application as fertilizer
• Extracellular component
– Excreted proteins
– Metabolic products – organic acids, alcohols
• Intracellular component
– Cytoplasmic or membrane proteins
– Lipids - biodiesel
Partly Science and Partly Art!
(Reference: Dr. Sridhar Viamajala for slides,
with some modifications by Dr. Ron Sims)
Cost determinants
• # of unit operations
– ↑ equipment and ↑ processing  ↑ cost
• Concentration of product at start of downstream
process
– Concentrating dilute products requires higher
throughput
• Purity and activity required
– Polishing steps are often more expensive
– Pharmaceutical products
– Analytical grade chemicals/enzymes
• Yield
Cost determinants (cont.)
Source: Doran PM, Bioprocess
Engineering Principles. 7th ed,
2002.
Dwyer Plot: Relationship between selling price and
concentration before downstream processing
Biotech products market
Reference: Textbook (Harrington, et al., Bioseparations Science and Engineering
Bioproduct categories
Bioproducts cont.
• Small biomolecules
– Primary metabolites: produced during growth
• Sugars
– Sucrose: sugarcane, sugar beets
– Fructose: by glucose isomerase
– Glucose: amylase treatment of starch
• Organic acids, alcohols, ketones
– Anaerobic fermentation
• Vitamins
– Organic synthesis
– Plant sources and microbial fermentation
• Proteins
– Highest commercial value - Pharmaceutical industry
– Produced from microbial, plant and animal cells
• Lipids
– Plants and microorganisms (algae)
– Products include steroids and biodiesel
Bioproducts cont.
– Secondary metabolites – produced during
stationary phase
• E.g. antibiotics such as penicillin
• Sources include fungi, bacteria, plant and animal tissues
Protein classification
Factors affecting protein
activity/stability
• Physical stability
– Temperature
• Mechanical stability
– Shear stress
– Pressure
– Surface tension
• Chemical stability
–
–
–
–
pH
Solvents
Chaotropic agents – break hydrophobic interactions
Detergents – for solubilizing cell membranes to purify membrane
bound protein
• Biological Attack
– Proteolysis
Source: Burgess R, Protein Purification in Protein Engineering, DL Oxender and CF Fox, eds., Alan R. Liss, Inc., 1987.
Protein properties determine
purification strategy
• Size and shape
– 3D structure, prosthetic groups
– Protein/enzyme activities must be preserved!!
• Charge and pI
– Net charge depends on pH
• Charge distribution
– Depends on protein folding and 3D structure
– Surface charge maybe different from overall charge
• Hydrophobicity
• Aggregation
– Reversible or irreversible
• Solubility
– Depends on all the above
• Density
• Ligand/metal binding
– Affinity properties
Source: Burgess R, Protein Purification in Protein Engineering, DL Oxender and CF
Fox, eds., Alan R. Liss, Inc., 1987.
Pre-Purification Steps
You know what protein to purify and you know all its properties – what
do you do next?
Step 1: Choose or Make a RICH source
• Host selection
– Procaryote
• Gram –ve (E. coli)
• Gram +ve (B. subtilis)
– Eucaryote
• Yeast
• Mammalian
• Plant
• Target location
–
–
–
–
–
–
Extracellular
Cytoplasmic
Periplasmic
Membrane bound
Organelle
Inclusion bodies
Pre-Purification Steps (cont.)
Host Selection
Source: Blanch HW and Clark DS. Biochemical Engineering. Marcel Decker, Inc., 1996
Pre-Purification Steps (cont.)
You know what protein to purify, you know all its properties and you have a source – what next?
Step 2: Develop an assay
•
Purity
–
Electrophoresis
•
PolyAcrylamide Gel Electrophoresis (PAGE)
–
–
•
Isoelectric focusing (IEF)
–
–
•
Separates based on isoelectric point
Varying pH, constant pore size
Concentration
–
UV absorption
–
Protein assay
•
•
280 or 254 nm
Bradford Method
–
–
–
•
•
Coomassie dye binds to Arginine and hydrophobic amino acids
Unbound dye is green and bound dye is blue (595 nm)
High Sensitivity
Lowry Method
–
–
–
–
Cu(II) in alkaline solution reacts complexes with protein
Protein-Cu(II) complexes react with Folin-Phenol reagent (phosphotungstic acid + phospomolybdic acid + phenol)
Product is blue and can be detected at 630 nm
Less sensitive than the Bradford method
Antibody-based Assays
–
–
–
–
–
•
Separates based on size/charge ratio
Constant pH, varying pore size
Protein-specific antibody binds to protein, (1º antibody)
(Protein+ 1º antibody) complex is reacted with a 2º antibody that carries a fluorescent molecule
Fluorescence can be visualized and/or quantified
Extremely sensitive method
If 2º antibody is an enzyme the method is called Enzyme-Linked ImmunoSorbent Assay (ELISA)
Activity
–
–
Activity assays with protein/enzyme-specific substrates
Can be done in-situ on proteins separated on a non-denaturing gel
Source: Garcia AA, Bonen MR, Ramirez-Vick J, Sadaka M, Vuppu A. Bioseparation Process Science. 1st ed., Blackwell Science, 1999
Stages of Downstream Processing
(Table 1.9)
Stage
Unit Operations
1.
Separation of insolubles
filtration, sedimentation,
extraction, adsorption
2.
Isolation of Product
extraction, adsorption,
ultrafiltration, precipitation
3.
Purification
chromatography,
crystallization, fractional
precipitation
4.
Polishing
drying, crystallization
Typical flow diagram of a protein
production facility
Source: Datar R and Rosen CG. Downstream Process Economics in Separation Processes in Biotechnology, Asenjo J ed., MarcelDekker, Inc., 1990
Typical flow diagram of a protein
production facility
Fermentation –
upstream processing
Source: Datar R and Rosen CG. Downstream Process Economics in Separation Processes in Biotechnology, Asenjo J ed., MarcelDekker, Inc., 1990
Typical flow diagram of a protein
production facility
Harvest and removal
of solids
Source: Datar R and Rosen CG. Downstream Process Economics in Separation Processes in Biotechnology, Asenjo J ed., MarcelDekker, Inc., 1990
Typical flow diagram of a protein
production facility
Primary recovery
Source: Datar R and Rosen CG. Downstream Process Economics in Separation Processes in Biotechnology, Asenjo J ed., MarcelDekker, Inc., 1990
Typical flow diagram of a protein
production facility
Secondary recovery
and polishing
Source: Datar R and Rosen CG. Downstream Process Economics in Separation Processes in Biotechnology, Asenjo J ed., MarcelDekker, Inc., 1990
Basic Principles of Engineering Analysis
Three principal ingredients of engineering analysis
1. Material Balance
Accumulation = inflow - outflow + amount produced - amount consumed
2. Equilibria
A+B=C
Keq = _[C]_
[A][B]
Keq = [CS] Partition coefficient when two phases are involved
[C]
3. Transport Phenomena (flux)
flux = coefficient x driving force
Example: Ohm’s law
Je = CE
Example: Diffusive flux (Fick’s first law) JD = -D dc/dx
Example: Flow through porous medium (Darcy’s Law) Jw=Lpdp
Process and Product Quality
Purity = _____amount of product__________________ (1.8.11)
amount of product + amount to total impurities
Specific activity = __units of biological activity__
mass
(1.8.12)
Yield = amount of product produced
amount of product in feed
(1.8.13)
Fold Purification = __purity at any stage in the process________
purity at the state of the purification process
Criteria for Process Development
Use of developing and evaluating a bioseparation process
Product purity
Cost of production as related to yield
Scalability
Reproducibility and ease of implementation
Robustness with respect to process stream variables
Route to Market
Section 1.9.3
GLP and cGMP
GLP = good laboratory practice
cGMP = current good manufacturing practice
IND = investigative new drug
“the process defines the product”