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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.