Downstream process modeling
Lecture 1
Mahesh Bule
Generalized View of Bioprocess
RAW MATERIALS
UPSTREAM PROCESSES
Inoculum
Preparation
Equipment
Sterilization
Media Formulation
and
Sterilization
BIOREACTOR - FERMENTER
Reaction Kinetics
and Bioactivity
Transport Phenomena
and Fluid Properties
Instrumentation
and Control
DOWNSTREAM PROCESSES
Separation
Recovery and
Purification
Waste Recovery,
Reuse and Treatment
THE BOTTOM LINE
REGULATION
ECONOMICS
HEALTH AND SAFETY
Typical Bioprocess Flow Sheet
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!
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
•
–
280 or 254 nm
Protein assay
•
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
2.
Isolation of Product
3.
Purification
4.
Polishing
filtration, sedimentation,
extraction, adsorption
extraction, adsorption,
ultrafiltration, precipitation
chromatography,
crystallization, fractional
precipitation
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., Marcel-Dekker, 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., Marcel-Dekker, 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., Marcel-Dekker, 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., Marcel-Dekker, 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., Marcel-Dekker, 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
Filtration process
• Filtration/centrifugation are the first steps after the
bioreactor to treat the fermentation broth
• Separate the water soluble from insoluble solids
(particulate matter, cells, cell fragments) present in the
fermentation broth.
• Removal of solids is important for all remaining
downstream separation and purification processes.
• Removal of solids prevent plugging of all adsorption and
ion-exchange packed columns and also prevents fouling
which causes increase in pressure drop and eventual
column failure.
Filtration of Fermentation Broth
Filtration is complicated by several factors specific
to biochemical processes:
• Nature, morphology and size of microbes.
• pH and viscosity of fermentation mixture (broth).
• Temperature history of broth.
• Contamination of broth by undesirable microbes.
• Nature of insoluble portion of residual substrate.
Filtration introduction
• Filtration may be defined as the separation of
solids from liquids by passing a suspension
through a permeable medium which retains
the particles.
Figure 1. Schematic diagram of filtration system
Types of filtration
1. Surface filters
2. Depth filters
1. Surface filters
used for cake filtration in which the solids are
deposited in the form of a cake on the up-stream
side of a relatively thin filter medium.
Figure2. Mechanism of cake filtration
2. Depth filters
used for deep bed filtration in which particle
deposition takes place inside the medium and cake
deposition on the surface is undesirable.
Figure 3. Mechanism of deep bed filtration
Filtration process considerations
• The fluid passes through the filter medium,
which offers resistance to its passage, under
the influence of a force which is the pressure
differential across the filter.
rate of filtration = driving force/resistance
Filtration process considerations
•The filter-cake resistance is obtained by multiplying the
specific resistance of the filter cake, that is its resistance
per unit thickness, by the thickness of the cake.
•The resistances of the filter material and pre-coat are
combined into a single resistance called the filter
resistance.
•It is convenient to express the filter resistance in terms
of a fictitious thickness of filter cake.
•This thickness is multiplied by the specific resistance of
the filter cake to give the filter resistance.
Filtration theory
Factors affecting filtration process
•
•
•
•
•
•
Pressure drop ( ∆P )
Area of filtering surface ( A )
Viscosity of filtrate ( v )
Resistance of filter cake ( α )
Resistance of filter medium ( Rm )
Properties of slurry ( μ )
Parameter correlation with filtration
rate
Parameter correlation with filtration
rate
-(P) or
Pressure
drop
rate of filtration = driving force/resistance
Filter cake ()
Filter medium (Rm)
Viscosity ()
Filtration process equations
Filtration process equations
Filtration process equations
Filtration process equations
Filtration process equations
Porosity (ε) and specific resistance (α)
in filtration
Porosity (ε) and specific resistance (α)
in filtration
Filter cake properties
Filter cake properties
Effect of compressible filter cake
Constant pressure
Effect of compressible filter cake
variable pressure
Effect of compressible filter cake
Constant flow rate
Average specific cake resistance
(αAVG)
Average specific cake resistance
(αAVG)
Average specific cake resistance
(αAVG)
Average specific cake resistance
(αAVG)
Filtration equipment's
The basic requirements for filtration equipment are:
• mechanical support for the filter medium
• flow accesses to and from the filter medium
• provision for removing excess filter cake.
Filtration equipment: (a) plate and frame press
(b) rotary vacuum filter (c) centrifugal filter
1. Plate and frame filter press
• In the plate and frame filter press, a cloth or mesh is
spread out over plates which support the cloth along
ridges but at the same time leave a free area, as large as
possible, below the cloth for flow of the filtrate.
• The plates with their filter cloths may be horizontal, but
they are more usually hung vertically with a number of
plates operated in parallel to give sufficient area.
• In the early stages of the filtration cycle, the pressure
drop across the cloth is small and filtration proceeds
at more or less a constant rate.
• As the cake increases, the process becomes more
and more a constant-pressure one and this is the
case throughout most of the cycle.
• When the available space between successive frames
is filled with cake, the press has to be dismantled and
the cake scraped off and cleaned, after which a
further cycle can be initiated.
• The plate and frame filter press is cheap but it is
difficult to mechanize to any great extent.
• Filtration can be done under pressure or vacuum.
• The advantage of vacuum filtration is that the pressure drop
can be maintained whilst the cake is still under atmospheric
pressure and so can be removed easily.
• The disadvantages are the greater costs of maintaining a
given pressure drop by applying a vacuum and the
limitation on the vacuum to about 80 kPa maximum.
• In pressure filtration, the pressure driving force is limited
only by the economics of attaining the pressure and by the
mechanical strength of the equipment
BAS stainless steel plate and frame filter press
Pressure leaf filters
Horizontal plate and frame filtration
system
Vertical plate and frame filtration
system
2. Rotary filters
• In rotary filters, the flow passes through a rotating
cylindrical cloth from which the filter cake can be
continuously scraped.
• Either pressure or vacuum can provide the
driving force, but a particularly useful form is the
rotary vacuum filter.
Rotary Vacuum Filter
A rotary vacuum filter is a continuous filter partially submerged
in the slurry.
• A drum is covered with a filter medium.
• Vacuum is applied to within the drum
• As the drum rotates, the solid constituent is separated by retained
on the porous medium
The liquid is drawn through the cake
into the inner filtrate pipes.
Each revolution consists of cake formation,
cake washing (if required),
drying and cake discharge.
Typical Flow Diagram of Rotary
Vacuum Filtration Process
http://www.solidliquid-separation.com/
VacuumFilters/vacuum.htm
http://www.komline.com/Products_Services/
Filtration/RotaryVac.html
Rotary Vacuum Filtration
Rotary Vacuum Filtration
3. Centrifugal filters
• Centrifugal force is used to provide the driving force in
some filters.
• These machines are really centrifuges fitted with a
perforated bowl that may also have filter cloth on it.
• Liquid is fed into the interior of the bowl and under
the centrifugal forces, it passes out through the filter
material.
Centrifugal filters
4. Air filters
• Filters are used quite extensively to remove
suspended dust or particles from air streams.
• The air or gas moves through a fabric and the dust
is left behind. These filters are particularly useful for
the removal of fine particles.
• The air passing through the bags in parallel. Air
bearing the dust enters the bags, usually at the
bottom and the air passes out through the cloth
• A familiar example of a bag filter for dust is to be
found in the domestic vacuum cleaner. Some designs
of bag filters provide for the mechanical removal of
the accumulated dust.