This Lecture
CHNG 3804
Bioseparations
Example Recovery of growth Hormon
After we have grown our biomass/made our
product how do we recover it?
In your other Chemical Engineering subjects you
have learnt about separation processes
What limitations are imposed by bioprocesses
Temperature
pH
GMP and cleaning
Waste minimisation
School Bioseparations Plant
Fermentation
Cell concentration
Cell disruption
Separation
and purification of
inclusion bodies
Solubilisation of
inclusion bodies
Refolding of protein
Biomass Separation
The first step of many bioseparation
processes is to separate biomass from the
fermentation broth:
Waste Water Treatment
Extra-Cellular Products
Intra-Cellular Products
Due to the large range of volumes/product
values a large range of techniques are
used
Biomass Separation Methods
Settling
Primary Method
Used with low value products waste water
Floatation
Dissolved Air Floatation (Waste Water Treatment)
Filtration
Widely used
Various Methods
Centrifugation
Filtration
School Bioseparations Plant
Plate filters
Continuous rotary-drum vacuum filter
A vacuum is pulled on a rotating drum
Liquid is sucked onto the drum and removed
by a scraper
Cross Flow Filtration
Use is increasing dramatically due to the
rapidly declining cost of membranes
Can use flat sheets or hollow fibres
Cross Flow Filtration
Cross Flow Filtration
Filtration Theory
Filtration Models
Flux through a filter tends to decline with
time
The shear in cross flow filtration reduces
this flux decline.
A number of models exist to explain this
Filter cake (Flux goes to zero)
Film (Flux reaches a minimum non-zero
value)
Flux
Film
Filter Cake
Time or Volume Filtered
Centrifugation
Centrifugation is used to separate
materials of different densities
Enables to use a force greater than gravity
Solution density can be varied to
selectively remove one component
Centrifugation
Batch
Lab or small scale
500 000g
Improve separation by increasing centrifuge
time
Continuous
For larger scale operation
Improve performance by reducing flowrate
Tubular Bowl Centrifuge
Axis of
rotation
Liquid
Overflow
r1
Particle
trajectory
r2
Feed Flow
Liquid
Surface
Simplest type of Centrifuge
Widely employed
Feed enters under pressure
through a nozzle at the bottom
As the bowl rotates particles
travelling upwards are spun
out and collide with the walls
of the of the bowl
Efficiency declines as solids
build up
13,000 to 16,000 g
More expensive than filtration
Disk Stack Centrifuge
Normal arrows Feed
Dashed - Light Product
Bold Heavy Liquid
Centrifugation Theory
ug =
p f 2
Dp g
18
Where
u g sedimentation velocity under gravity
p density of particle
f density of liquid
Feed enters through the top
Common in bioprocessing
Manual, continuous and
intermittent solids removal are all
possible
Small clearances between the
conical sections
5000 15,000 g
Requires a density difference of
> 0.01-0.03 kg/m3
Minimum particle 0.5m
Centrifugation Theory
uc =
p f 2 2
D p r
18
Where
uc is the velocity in the centrifuge
is the angular velocity (rad/s)
viscosity of liquid
r is the radius
D p particle diameter
Z factor relates force in centrifuge to gravity
g gravitational acceleration
Z=
2r
g
Industrial centrifuges have Z factors from 300 to 16,000.
For small laboratory centrifuges Z may be up to 500,000.
Centrifugation Theory
The performanc e of different centrifuge s can be related
by the Sigma Factor
=
Q
2u g
Physically the Sigma Factor represents the cross - sectional
area of a gravity settler with the same performanc e as the centrifuge
If two centrifuge s perform with equal effectiven ess
Q1 Q 2
=
1 2
Tubular Bowl
=
2b
(3r
2
2
+ r12
2g
Where b is the length of the bowl
r1 is the radius of the liquid surface
r2 is the inner radius of the bowl
As r1 r2
=
2 2br 2
g
Techniques for Cell Disruption
Grinding with abrasive
High speed agitation
High Pressure homogenisation (widely used)
Widely used in the dairy industry, food industry and for making
emulsions.
Typically Operate at ~50MPa, may require multiple passes
Pressure is let down through two valves
Generally need cooling so that products are not denatured
Flow rates from 1L/min upwards
Ultrasound
Non-mechanical methods
Osmotic shock
Freezing and thawing
Enzymatic digestion of cell walls
Treatment with solvents and detergents
Centrifugation Theory
2 2 (N 1) 3 3
r2 r1
3g tan
Where
N is the number of discs
r2 is the outer radius of the disc
r1 is the inner radius of the disc
is the half cone angle of the disc.
Cell Disruption
Downstream processing of fermentation broths usually
begins with separation of cells by filtration or
centrifugation.
Next step depends on location of the desired product.
For ethanol, citric acid and antibiotics which are excreted
from cells, product is recovered from the cell-free broth.
Biomass is discarded or sold as a by-product.
For products such as enzymes, recombinant proteins
which remain in the biomass, cell disruption must be
carried out to release the desired material.
Cell Disruption
Homogenisation
High Pressure Piston pump
Widely used in the dairy industry, food
industry and for making emulsions.
Typically Operate at ~50MPa, may require
multiple passes
Pressure is let down through two valves
Generally need cooling so that products are
not denatured
Flow rates from 1L/min upwards
Homogeniser
Homogenisation
Hetherington et al., (1971) modelled the release of
soluble protein from homogenized yeast.
They found that after N passes, the release of protein
(Rp) could be described by
ln
1
= NP a
1 Rp
Where P was the pressure, a and were constants.
Sauer et al., (1989) modified this equation, for use with
E.coli, by the addition of an exponent (b) to the number
of passes N, giving Equation
ln
Homogenisation
For E.coli disruption a
similar model can be
used, where D is the
disruption.
Typical parameter
values where P is in
MPa
Cell Disruption
1
ln
= N b P a
1 D
Microfluidisation
Smaller High Pressure device
Typically uses an air powered motor (Noisy)
Different method of causing the cells to break
Ultrasonication
Parameter
Value
1.4
0.95
9.7 x 10-4
Solubilisation and Refolding
Proteins produced using Recombinant
bacteria are typically not in their correctly
folded form.
The most common method used to fold
proteins correctly is to
Solubilise the protein
Then refold it
1
= N b P a
1 Rp
Uses ultrasonic waves to disrupt cells
Useful at small scale
Enzymatic
Lysozyme is an enzyme present in tears and saliva
that can breakdown cell walls
Useful for analytical applications - Electrophoresis
Solubilisation
The high concentration of Urea helps to denature the
Protein
The -mercaptoethanol breaks the S-S bonds between the
side chains
Solubilisation ~ 2hrs
A centrifugation step can be added to remove insoluble
components
Species
Concentration
pGH
10 mg/mL
Urea
8M
Tris Base
10 mM
-mercaptoethanol
100 mM
Refolding
Lane
6
10
MW
(kDa)
94
After a protein has been
solubilised it needs to
refolded
GSH and GSSG are used
to break and reform S-S
bonds
It is important to add the
protein slowly
Typically 2-3 days at 4oC
67
Species
Concentration
pGH
0.9 mg/mL
Urea
2M
Tris Base
10 mM
pH
GSH
0.1 mM
GSSG
0.01 mM
Sodium
Azide
0.02%
43
30
20
14
1 is the marker, 2 the solubilised suspension, 3 solubilised supernatant, 4 the
pellet after centrifugation of the solubilised inclusion body.
5 & 6 samples from the refolding under reduced conditions.
7 is blank, lane 8, 9 & 10 are samples from the refolding under non-reduced
conditions.
Solvent Extraction
Familiar from Mass Transfer
Used in Penicillin recovery (Organic)
Small scale
Separating Funnel
Large Scale
Column
Organic phases are unsuitable for proteins and
sensitive bio-polymers
Two phase aqueous systems are used instead
Adsorption
Adsorption is a surface phenomenon
Four types
Exchange
Physical
Chemical
Non-Specific
Scale up methods not well defined
Ideal Adsorbent has a high surface area to
volume ratio
Chromatography
Cleaning
Separation procedure based on differential
migration
Gas
If we are producing/separating a product
produced by biological systems it is important to
be able to
Use for analysis
Adsorption
Analysis and Final Product Purification
Two liquids
Normal or Reverse Phase
Sterilise
Clean
Sanitise
This has implications for
Materials Stainless Steel
Valves
Piping design Self Draining
Cleaning
May design for Clean in Place (CIP)
Cleaning agents
Steam
Hypochlorite
P3-Oxonia (A mixture of Peracetic Acid and
Hydrogen Peroxide)
It may be necessary to validate cleaning
by taking swaps and plating etc.
Summary
The separation and recovery of products is
an essential part of any bioprocessing
operation.
Many Chemical Engineering Operations
are used.
Bioprocesses can limit the types of
operations used.
