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1
CHNG 3804CHNG 3804
Bioseparations
This LectureThis Lecture
• 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
Example Recovery of growth Example Recovery of growth HormonHormon
Fermentation
Cell disruption
Cell concentration
Separationand purification ofinclusion bodies
Solubilisation ofinclusion bodies
Refolding of protein
School School BioseparationsBioseparations PlantPlant
Biomass SeparationBiomass Separation
• The first step of many bioseparationprocesses 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 MethodsBiomass 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
2
FiltrationFiltration
• 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
School School BioseparationsBioseparations PlantPlant
Cross Flow FiltrationCross Flow Filtration Cross Flow FiltrationCross Flow Filtration
Filtration TheoryFiltration Theory
• 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)
Filtration ModelsFiltration Models
Filter Cake
Film
Time or Volume Filtered
Flux
3
CentrifugationCentrifugation
• 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
CentrifugationCentrifugation
• 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 CentrifugeTubular Bowl Centrifuge
• 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
r1
r2
Feed Flow
Liquid Overflow
Liquid Surface
Particle trajectory
Axis of rotation
Disk Stack CentrifugeDisk Stack Centrifuge
• 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 0.5µmNormal arrows – FeedDashed - Light ProductBold – Heavy Liquid
Centrifugation TheoryCentrifugation Theory
onaccelerati nalgravitatio
diameter particle liquid of viscosity
liquid ofdensity
particle ofdensity
gravityunder ty ion velocisedimentat Where
182
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u
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f
p
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µρρ −
=
Centrifugation TheoryCentrifugation Theory
gr
Z
r
u
rDu
c
pfp
c
2
22
gravity tocentrifugein force relatesfactor Zradius theis
(rad/s)locity angular ve theis
centrifuge in the velocity theis Where18
ω
ω
ωµρρ
=
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Industrial centrifuges have Z factors from 300 to 16,000.For small laboratory centrifuges Z may be up to 500,000.
4
Centrifugation TheoryCentrifugation Theory
2
2
1
1 QQesseffectiven equal with perform scentrifuge twoIf
centrifuge theas eperformanc same th thesettler wigravity a of area
sectional-cross therepresentsFactor Sigma thePhysically
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Factor Sigma by therelated becan scentrifugedifferent of eperformanc The
Σ=
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=Σgu
Q
Centrifugation TheoryCentrifugation Theory
( ) ( )
disc. theof angle cone half theis
disc theof radiusinner theis
disc theof radiusouter theis discs ofnumber theis N
tan312
1
2
31
32
2
θ
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r
r
Where
rrg
N −−=Σ
Tubular BowlTubular Bowl
( )
gbr
r
r
b
rrg
b
22
21
2
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2
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r r As
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32
πω
πω
=Σ
≈
+=Σ
Cell DisruptionCell 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.
Techniques for Cell DisruptionTechniques 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
Cell DisruptionCell 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
5
HomogeniserHomogeniser HomogenisationHomogenisation
• 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
• Where P was the pressure, a and α 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
a
p
NPR
α=��
�
�
��
�
�
−11
ln
ab
p
PNR
α=��
�
�
��
�
�
−11
ln
HomogenisationHomogenisation
• For E.coli disruption a similar model can be used, where D is the disruption.
• Typical parameter values where P is in MPa
ab PND
α=��
���
�
−11
ln
9.7 x 10-4α
0.95b
1.4a
ValueParameter
Cell DisruptionCell Disruption
• Microfluidisation– Smaller High Pressure device– Typically uses an air powered motor (Noisy)– Different method of causing the cells to break
• Ultrasonication– 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 and RefoldingSolubilisation 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
SolubilisationSolubilisation
• The high concentration of Urea helps to denature the Protein
• The β-mercaptoethanol breaks the S-S bonds between the side chains
• Solubilisation ~ 2hrs• A centrifugation step can be added to remove insoluble
components
100 mMβ-mercaptoethanol
10 mMTris Base
8 MUrea
10 mg/mLpGH
ConcentrationSpecies
6
RefoldingRefolding
• 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 4oC0.02%
Sodium Azide
0.01 mMGSSG
0.1 mMGSH
8pH
10 mMTris Base
2 MUrea
0.9 mg/mLpGH
ConcentrationSpecies
MW(kDa)
94
67
43
30
20
14
Lane1 2 3 4 5 6 7 8 9 10
•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 ExtractionSolvent 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
AdsorptionAdsorption
• 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
ChromatographyChromatography
• Separation procedure based on differential migration
• Gas– Use for analysis
• Adsorption– Analysis and Final Product Purification
• Two liquids– Normal or Reverse Phase
CleaningCleaning
• If we are producing/separating a product produced by biological systems it is important to be able to– Sterilise– Clean – Sanitise
• This has implications for– Materials – Stainless Steel– Valves– Piping design –Self Draining
7
CleaningCleaning
• 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.
SummarySummary
• The separation and recovery of products is an essential part of any bioprocessingoperation.
• Many Chemical Engineering Operations are used.
• Bioprocesses can limit the types of operations used.