Kein Folientitel

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Encapsulation as a technology for removing,
dosing or measuring in a wide range of fields and
environments
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School of Biotechnology Bioprocess Engineering
Group
This is a continuously evolving scheme with
different areas joining or leaving the circle of
interests
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Integrated bioprocessing

• What do we mean?
•  Bioprocesses which have been designed by taking
  into account the whole process from USP to DSP
  and in particular validation issues and the
  quality of the desired product (holistic
  approach). This usually involves incorporation of
  key on-line measurement/ control and product
  removal systems or in-situ product recovery
  systems (ISPR).

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Integrated bioprocessing
In the context of this talk Use of novel
encapsulation technologies for achievement of
Increased productivity through high cell density
culture e.g mammalian cells
In-situ- product recovery (ISPR) for increased
productivity through removal of product
inhibition in fermentation processes Capsular
perstraction
ISPR for increased productivity through removal
of toxic compounds in water treatment (CP)
Reactive capsular perstraction enzyme
encapsulation- combined bioconversion with
product separation - reactive chromatography
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But first- what are (micro-) capsules?

• Multitude of excipients for wall polymer
• In general must be difference in either
  solubility and/ or viscosity between core
  material and wall polymer
• Choice depends on function e.g. implant, drug
  delivery, probiotic delivery i.e.
  biocompatibility, stability and
• Capsule production method
• Common polymers calcium alginate
  alginate/poly-L-lysine cellulose sulphate/
  PDADMAC polyacrylamide chitosan, k-carageenan,
  block co-polymers etc.
• Two types of core
• Aqueous (hydrophilic) core
• Lipophilic core
• Cores may be solid (immobilization) or liquid
  (encapsulation)
• Only liquid core capsules will be dealt with here

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Second What is the application?

• Medical
• Implants (bioartificial pancreas, liver, drug
  delivery)
• Pharmaceutical
• Drug production, cell culture, drug delivery/
  formulation
• Food and beverages
• Flavour and aroma delivery, fermentation, taste-
  masking, neutraceuticals, probiotic delivery
• Extraction
• Product recovery (ISPR)
• Pesticide/ herbicide removal
• (Wyss et al., Biotechnol. Bioeng. 2004 87,
  734-742)
• Bioconversions
• whole cell and enzyme- catalyzed reactions (Wyss
  et al 2004 Biotechnol. Bioeng.)

Choice of polymer depends on application
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Third What are the desired properties?
(Quantitative methods)

• Size
• Size distribution
• Mechanical strength
• Molecular weight cut- off
• Liquid/ solid core
• Stability (dissolve with time, long- term, etc.)
• Surface properties (neutral, charged,
  hydrophobic)
• .etc.

Choice of polymer also depends on desired
properties
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Fourth Determine Type of excipient (polymers)

• Polyelectrolytes
• Ca- alginate, alginate-polylysine, cellulose
  sulphate-polydiallyldimethylammonium chloride
  (PDADMAC), chitosan, CMC etc.
• Cross- linked polymers
• Polyacrylamide, alginate-PGA, PVA, block
  copolymers etc.
• Thermal polymerization
• Collagen, gelatin, agarose, gum arabic,
  k-carrageenan, waxes etc.
• Photopolymerization
• The choice of polymer(s) depends on the
  application, properties required and production
  method

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Fifth Choice of production technique
Production techniques for liquid- core capsules

• Emulsion-based techniques
• Melt encapsulation
• Emulsification/ internal gelation
• Phase separation-coacervation
• Interfacial polymerization
• In-situ polymerization
• Spray drying
• Spray chilling
• Spray freezing

• Fluidized-bed coating
• Atomization
• Centrifugal extrusion
• Jet cutting
• Electrostatic droplet generation
• Prilling
• Etc. etc.

Important reproducible, spherical, monodisperse
size distribution
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So how do we make microcapsules of defined size,
shape and properties..?
Solid core (beads)
Liquid core (capsules)
or
Diameter of microbeads 200 mm Standard deviation
of sample 2.9
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using a vibrating jet extrusion technique
To make solid core capsules (beads)
Flow rate up to 2 l/h
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.but for this exercise we need liquid- core
capsules Prilling
Production of liquid core capsules of sunflower
oil with calcium alginate (1.5) wall
Concentric nozzle system
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Case study 1 Animal cell encapsulation
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Methods currently used for raising cell density/
productivity

• Membrane reactors e.g. hollow fibres
• Perfusion processes with cell recycle devices
• Immobilization
• Encapsulation

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Why cell encapsulation?

• Animal cells sensitive to mechanical shear forces
  (aeration, agitation and pumping)
• Anchorage-dependent cells require a high
  attachment surface area
• Continuous suspension cell cultures limited by
  low specific productivity (1-10 pg cell-1 h-1)
  and maximum cell density (1- 5 ? 106 cells mL-1)
• Higher cell densities (organ-level densities 1-3
  ? 108 cells mL-1) and productivities achieved by
  cell recycling or immobilization/ encapsulation

Capsule size should be lt 600mm diameter to avoid
O2 and nutrient limitation
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Advantages of cell encapsulation

• Avoids problems associated with immobilization on
  carriers (cell desorption, cell growth on
  surface, increasing particle size with time,
  shear at surface etc.)
• No aggregation
• Suitable for all cell types- suspension and
  anchorage
• Shear protected
• Molecular weight cut-off of membrane allows
  selective recovery of proteins- capture step
• Cell- free product- no separation step required
  in DSP
• Constant size- no diffusional limitations
• Potentially allows for production in absence of
  growth, when colonization of capsule core
  complete.

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Animal cell encapsulation
CHO cells secreting human secretory component
(hSC)
3 days
12 days
0 days
PGA, propylene-glycol-alginate
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Encapsulated CHO cells
800 mm capsules 1.5 litre bioreactor
Capsule Medium volume 115
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Conclusions
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Case study 2 Microcapsules in bioprocess
environments

• Capsular perstraction for
• In- situ product recovery (ISPR)
• from microbial cultures

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Inhibition by 2-phenylethanol
Complete growth Inhibition by 3.8 g/l 2-
phenylethanol
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Possible solution to continuously recover
product Two-Phase extractive bioconversion

Problems
Note high levels of organic phase
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Two-Phase extractive bioconversion
3.8 g/l
2.1 g/l
Organic phase inhibition synergistic effects of
solvent and 2-phenylethanol
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Solvent- containing alginate capsules
liquid-core capsules
Manufacturing of solvent core alginate capsules
via prilling
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Capsular Perstraction (Internal)
Note low levels of organic phase
Higher concentrations possible simply by
increasing number of capsules from 6 to 50
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Conclusions
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Case study 2 Microcapsules in the environment

• Capsular perstraction
• Removal of hydrophobic organic pollutants (HOPs)
  and persistent organic pollutants (POPs) from
  water using liquid- core capsules

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Problem?

• HOPs and POPs are often extremely toxic compounds
  that find their way into water courses
• The concentration of these compounds is very low
  therefore difficult to detect.
• Possible to degrade some of these compounds
  biologically but concentration either too low to
  sustain growth/ viability or too toxic
• All treatment processes involve biological,
  chemical, electrochemical or adsorption
• But must first pre-concentrate the pollutants-
  how?

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Log Kow values for some important HOPs
Log Kow Log (Coctanol/ Cwater) HOP
hydrophobic organic pollutant BCF
Bioconcentration factor
US EPA-822-R-93-007 (1993) Great Lakes Water
quality Initiative Criteria Documents for the
Protection of Wildlife
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What type of compounds can be extracted? e.g.
BTEXs
Solubility in water may be high but if Log Kowgt0
then more soluble in oil than water so can
separate by extraction/ perstraction
Perstraction extraction in which organic solvent
(e.g. oil) separated from water by membrane
Note Log Kow Log (Coctanol/ Cwater)
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Perstraction
Environmental applications e.g. water treatment
Pesticide/herbicide/PCB/dioxin/BTEX etc. logP gt
1 (usually very dilute i.e. ppm)
Pollutant concentrates upto 106 fold
Aqueous phase
Hydrogel membrane
What to do with capsules full of oil/ pollutant?
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Extraction of pesticides/ herbicides using
liquid-core capsules (perstraction)
2,4-D
atrazine
methylparathion
ethylparathion
The rate of extraction depends on capsule size,
number and core material (logP)
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Perstraction
Back extraction with simultaneous microbial
breakdown
Pollutant diffuses into water to saturation
concentration (non-toxic)
Pollutant concentrated upto 1010 fold
Pollutant degraded
Aqueous phase
Microbial culture (e.g. Pseudomonads capable of
degrading pollutant but pollutant toxic at high
concentration)
Hydrogel membrane
Could also incinerate capsules have high
calorific content
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Recycling of microcapsules through microbial
breakdown of atrazine
No inhibition with atrazine capsules
Inhibition by atrazine in solution above 100mg/L
Inhibition by dibutyl sebacate
Growth rate of Pseudomonas sp. ADP as a function
of atrazine concentration
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.when atrazine encapsulated- no inhibition
100 mg/L atrazine
200 mg/L atrazine
400 mg/L atrazine
Growth rates linear due to being limited by rate
of atrazine exo-diffusion from capsules (atrazine
is sole N-source)
Growth
Growth of Pseudomonas sp. ADP as a function of
atrazine concentration in 12ml liquid-core (DBS)
capsules
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Perstraction
Use capsule directly as biosensor to detect
presence of polluant
Pollutant concentrated upto 1010 fold
chromogen
Y
Y
Y
Y
Y
Y
Y
chromophore
Aqueous phase
Activated antibodies or chemical reaction?
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Case study 3 Microcapsules in bioconversion
reactions

• Reactive capsular perstraction
• or
• enzyme adsorption/ immobilization on liquid- core
  capsules

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Reactive perstraction
E
E
E
Enzyme
E
E
Hydrogel membrane
E
E
E
Aqueous core in organic solvent
Other possibilities
Aqueous core in aqueous solvent
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Reactive perstraction. Adsorbed/covalently bound
lipase
First immobilized versus free enzyme
System chosen due to simplicity in following
reaction (titration of propionic acid)
Core hexadecane
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Proprionic acid production as function of time
(hydrolysis of tripropionin)
Complete hydrolysis of 1 moleTP should yield
3mole proprionic acid
Demonstrates product inhibition
1.6 mM
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Specific activity of lipase as function of
tripropionin concentration
Immobilized lipase higher specific activity
Adsorbed lipase
Free lipase

• - longer stability
• - shift in pH optimum
• increased temperature
• stability (not shown)

ro VmS/Km S (1S/Kis)
Fitted with Michaelis- Menten kinetics with
substrate inhibition
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Reactive perstractionAdsorbed/covalently bound
lipase
Second demonstration of ISPR and reduction in
product inhibition
(almost insoluble in hexadecane at pH lt7.5)
Nitrophenyl laurate and lauric acid highly
soluble in hexadecane at pH 7.5
Core hexadecane
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Hydrolysis of nitrophenyl laurate by free lipase
in presence of liquid core capsulesi.e. with
perstraction
Hydrolysis of nitrophenyl laurate (NL partitions
into both aqueous and core phases but no effect
on hydrolysis rate)
AP nitrophenyl laurate
AP nitrophenol
OP lauric acid
Nitrophenol in aqueous phase
Lauric acid formed in capsule and aqueous
phase slowly extracted from AP into capsule core
AP lauric acid
OP nitrophenyl laurate
AP aqueous phase OP organic (hexadecane) core
phase
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Hydrolysis of nitrophenyl laurate by lipase
immobilized on liquid core capsulesi.e. with
reactive perstraction
Hydrolysis of nitrophenyl laurate (NL partitions
into and core phases but no effect on hydrolysis
rate)
500 mg capsules
AP nitrophenyl laurate
OP lauric acid
AP nitrophenol
Nitrophenol in aqueous phase
Lauric acid formed in capsule core phase only
since lower immobilized enzyme activity means
that process was reaction limited and not
diffusion limited
OP nitrophenyl laurate
AP aqueous phase OP organic (hexadecane) core
phase
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Conclusions

• Significant new advances being made with respect
  to integrated bioprocessing
• New capsule technologies being developed, such as
  high cell density culture, in-situ product
  recovery, simultaneous biocatalysis and product
  recovery.
• Encapsulation may play an important role in
  bioprocess development and optimization
• These advances should result in significant
  improvements in product concentration,
  productivity, process stability, reduced process
  volumes and reduced production costs.
• Bioencapsulation has a great future in many areas
  of bioprocessing

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Conclusions

• Encapsulation as a technology for removing,
  dosing or measuring in a wide range of fields and
  environments

Dosing drug delivery, implants, microbial
nutrient toxic supply
Measuring microsensors
Environments Human body, bioreactors, water
treatment plants etc.
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Future

• Immobilized microbial cells for high cell density
  bioconversions
• Immobilized enzymes for bioconversions e.g.
  cellulases, amylases, galactosidases etc.
• Immobilized enzymes for reactive capsular
  perstraction e.g. lipases, penicillin acylase,
  cephalospirin acylase etc. leading to reactive
  chromatography
• Capsules for ISPR and water treatment
• Capsules for metal recovery, microbial separation
  and antibiotic effects
• Drug and flavour delivery
• Development of high- throughput screening
  techniques
• Development of novel sensors based on micro- and
  nano-capsules
• Targetted drug delivery

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This leads to nanocapsules
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Nanocapsules (oleosomes)General data

• Structure of oil storage in oleaginous plant
  seeds.
• TAG matrix surrounded by a monolayer of
  phospholipids and embedded proteins (oleosins).
• 0.1 mm lt Ø lt 2.5 mm

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Oleosins Structure/function, properties

• Specific to seed NC (and pollen), where it is
  very abundant (up to 20 of the total seed
  proteins)
• Structure (remains to be fully elucidated)
• Highly conserved hydrophobic core (likely to be
  the longest naturally occurring hydrophobic
  sketch) of not well defined II structure.
• Amphipathic C- and N- terminal domain structure
  in a-helix.
• Properties
• Poorly to soluble
• Oligomerise in solution
• Alkaline pI (pI?9)
• Mw ranging from 15 to 24 kDa following species
  (generally two isoforms pro species)
• Function
• Stabilisation of NC (prevent coalescence)
• Binding site for lipase
• Regulate the size of NC
• Enables the seeds to withstand dehydration

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Nanocapsule PurificationFat pad
Flotation on density gradient ? we have OB

• Microscopy
• Stability
• Proteins

0.1 Tween 80 washing
2 M NaCl washing
9 M Urea washing
Washed nanocapsules
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Flocculation assays

• Jar Test
• assay

Introduction
OB Purification
Objectives
Conclusions
Summary
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Preliminary experiments

• NC extract show high flocculant activity
• The activity is variable following species
• Ca2 and sonication have a great effect

• This raises questions NC have a pI ? 6
• ? Why do the NC preparation show
  coagulant/flocculant activity ?

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Proteinase K model

• Nanocapsule proteins and other associated
  proteins ( )

Targetted drug delivery
Proteins/polysaccharides
Proteinase K
Flocculation activity disappears
Introduction
OB Purification
Objectives
Flocculation
Conclusions
Summary