Bio Separation

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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

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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

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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

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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.

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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


• 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

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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=��

�

�

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�

�

−11

ln

ab

p

PNR

α=��

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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


• 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

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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

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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.

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Bio Separation