As presented at the CRS Annual Meeting & Exposition 2018 by Steve Thomas, Oliver Batley and Mara Tavares (Cambridge Consultants) and Justyna Klimczac and Elżbieta Górecka (Proteon Pharmaceuticals)

1. 23 July 2018 P2777-P-024 v1.0
Steve Thomas, Oliver Batley, Mara Tavares – Cambridge Consultants Ltd.
Justyna Klimczac, Elżbieta Górecka – Proteon Pharmaceuticals S. A.
CRS Annual Meeting & Exposition 2018
ENCAPSULATION FOR INDUSTRIAL APPLICATIONS
A NOVEL, CONTINUOUS
MICROENCAPSULATION PROCESS FOR
INDUSTRIAL APPLICATIONS

2. 23 July 2018 P2777-P-024 v1.02
 Excellent area to utilise a wide range of the
skills at our disposal
 Novel processes by combining off-the-shelf
chemistry with innovative engineering
 In-house biological capability provides unique
offering when combined with chemistry, fluids,
electronics and software
 Offerings from technology landscaping
through to feasibility testing and delivery of
production process equipment
Our interest in microcapsules
MICROENCAPSULATION
APPLICATION &
EMBEDDED SOFTWARE
MECHANICAL
SENSORS
USER
INTERFACES
AUTOMATION AND
PROCESS DESIGN
MICRO & MACRO
FLUIDICS
CHEMISTRY
MOLECULAR
BIOLOGY

3. 23 July 2018 P2777-P-024 v1.03
 Agriculture
– Agricultural chemicals and crop spraying
– Deployment of biologicals e.g. Plant
Growth Promoting Bacteria (PGPB)
 Industrial
– Industrial processes and chemical
manufacture
– Smart coatings – e.g. anti fouling
– Smart materials – self healing, thermal
management
 Food, Beverage and Consumer
– Flavour and fragrance
– Vitamins and probiotics (Nutraceuticals +
Personal care)
 MedTech
– Drug delivery
Areas of particular interest
MICROENCAPSULATION

4. 23 July 2018 P2777-P-024 v1.04
 Proteon Pharmaceuticals produce
bacteriophage cocktails for the treatment of
salmonella and other pathogens in livestock
and poultry
 Initially developed as a liquid food/water
additive they have also modified it as an
encapsulated form for addition to dry feed
 Approached Cambridge Consultants looking
for a manufacturing process to produce
commercial volumes of their products for
testing and sale
Case Study: Bacteriophage
Encapsulation for Proteon
Pharmaceuticals

5. 23 July 2018 P2777-P-024 v1.05
 Bacteriophage cocktail may be used as an
alternative to antibiotics for the treatment of
salmonella in poultry
 Bacteriophage inject viral genome into
bacteria cell
– Facilitates replication of bacteriophage
– Results in death of host cell
 Unlike antibiotics, bacteriophages are highly
specific and do not harm natural microflora of
the animal being treated
 Because of this different mechanism of action,
bacteriophage cocktails may be used to fight
antibiotic-resistant bacteria
About Bacteriophage
CASE STUDY: BACTERIOPHAGE ENCAPSULATION

6. 23 July 2018 P2777-P-024 v1.06
 Scale up of process to enable production of
several kg quantities of microcapsules
 Process must be relatively low pressure
– High pressure ‘kills’ bacteriophage
 Suitable for sterilisation operation and
subsequent sterilisation
 Compact system for current lab facilities
 Process suitable for downstream expansion to
100 kg quantities
Requirements
CASE STUDY: BACTERIOPHAGE ENCAPSULATION

7. 23 July 2018 P2777-P-024 v1.07
 Low volume batch process
 Based around hardening polymer solution by
exchange of 1+ for 2+ counter ions
– Prepare polymer solution with bacteriophage
– Emulsify polymer solution in vegetable oil to
form water-in-oil emulsion
– Add solution of exchange ions to solidify
aqueous droplets into microcapsules
– Divide into batches and centrifuge to
separate out microcapsules from emulsion
Existing Process
CASE STUDY: BACTERIOPHAGE ENCAPSULATION

8. 23 July 2018 P2777-P-024 v1.08
 Process can be broken down into two key
stages
– Emulsion formation to define capsule size
– Mixing with hardening buffer to solidify
capsules
 Size distribution of microcapsules controlled
by first stage
 Second stage important to ensure separation
of capsules and control physical properties
Process Breakdown
CASE STUDY: BACTERIOPHAGE ENCAPSULATION
Base Solution
Aqueous polymer
solution +
Bacteriophage
Emulsify
Disperse to form Water-
in-Oil emulsion
Harden
Introduce 2+ ions
Define capsule size
Solidify and
ensure separation

9. 23 July 2018 P2777-P-024 v1.09
 Key process is emulsification
– Current method forces batch processing
 CC have worked with a number of flow mixing
technologies for a variety of rheologies
– Mayonnaise, moisturiser, cappuccino
 Allow control of emulsification
– Dimensions of Flow system
– Rate of flow of aqueous solution and oil
 Potential for in-line monitoring and close-loop
operation
 Choose device geometry to match rheological
properties of solutions
– Vortex mixer over channel mixers
 Hardening stage less critical but must ensure
intimate mixing and maintain capsule separation
Engineering Solution
CASE STUDY: BACTERIOPHAGE ENCAPSULATION

10. 23 July 2018 P2777-P-024 v1.010
 Basic system consists of pumped reservoirs
and mixing chambers
 First chamber: Controlled emulsification
 Second chamber: Mixing with exchange ion
solution
 Pump speed and timing controls ratios of
ingredients in reactions and final mix
Continuous Encapsulation System
CASE STUDY: BACTERIOPHAGE ENCAPSULATION
Emulsion
formation
Vegetable oil
Exchange Ion
Solution
Bacteriophage in
polymer solution
Capsule
hardening
Capsule
suspension

11. 23 July 2018 P2777-P-024 v1.011
 Benchtop process reproduced to yield similar microcapsule results
 Capsule dimensions dependent on stirring time, speed temperature
 Typically produces bimodal or trimodal distribution of sizes
 Process must produce capsules which are small enough to blend with food
but not so small that they form dust
 Able to demonstrate equivalent results with and without bacteriophage
present
– Process development can be performed with dummy solution
Process Reproduction
CASE STUDY: BACTERIOPHAGE ENCAPSULATION

12. 23 July 2018 P2777-P-024 v1.012
 Process development requires thorough
characterisation
 Replication and improvement over benchmark
process
 Testing performed against a number of metrics
– Capsule size distribution
– Bacteriophage plaque formation
– Yield and encapsulation efficiency
Testing
CASE STUDY: BACTERIOPHAGE ENCAPSULATION

13. 23 July 2018 P2777-P-024 v1.013
 Size distribution of capsules from continuous
and batch processes characterised using
Malvern Mastersizer system
 Graphs opposite show typical behaviour from
a similar system
– Mineral oil encapsulated in nylon shell
– Sebacoyl Chloride in oil reacting with
Hexamethylenediamine in water
– Interfacial polymerisation
 Same test methodology used to optimise
equipment and process for bacteriophage
encapsulation
Testing: Capsule size distribution
MEASUREMENT OF MICROCAPSULE SIZE DISTRIBUTION

14. 23 July 2018 P2777-P-024 v1.014
 Testing of bacteriophage activity through
encapsulation process is performed by assay
against target pathogen
 Test used SalmonellaG003 strain provided by
Proteon Pharmaceuticals
 Class II pathogen
 Closely related to Salmonella Gallinarum
 Pathogenic to poultry but not humans
Testing: Bacteriophage Activity
CASE STUDY: BACTERIOPHAGE ENCAPSULATION

15. 23 July 2018 P2777-P-024 v1.015
 Microcapsules are re-dissolved for plate assay
 Bacteriophages are enumerated by agar
overlay plate method
– Salmonella Gallinarum cultured as host
medium
– Serial dilutions and plaque counting
Testing: Plaque formation assay
CASE STUDY: BACTERIOPHAGE ENCAPSULATION

16. 23 July 2018 P2777-P-024 v1.0
 Custom software to count plaques
 Variable contrast thresholds
 Classifies plaques by size distribution – distinct populations
classified by plaque size bin
 Feasibility for future automation of assay procedure
Automated Plaque Enumeration
CASE STUDY: BACTERIOPHAGE ENCAPSULATION

17. 23 July 2018 P2777-P-024 v1.017
 Enumeration and conversion to equivalent
dilutions shows slight loss of phage activity
from both the batch and continuous
encapsulation processes
 More work on process optimisation for both
capsule size distribution and bacteriophage
yield
Phage Enumeration Results
CASE STUDY: BACTERIOPHAGE ENCAPSULATION
1.0E+00
1.0E+01
1.0E+02
1.0E+03
1.0E+04
1.0E+05
1.0E+06
1.0E+07
Polymer Solution +
bacteriophage
Batch Process Continuous Process
EquivalentPFU/ml
BA - Polymer +
Bacteriophage
CC - Continuous Process
PP - Batch Process

18. 23 July 2018 P2777-P-024 v1.0
 Developed novel continuous microencapsulation system
 Demonstrated system for production of encapsulated bacteriophage cocktail for treatment of salmonella in poultry and
livestock
 Characterised microcapsule size distribution for continuous and batch versions of process
– Capsule size tuneable via liquid flow rate and chamber dimensions
 Characterised encapsulation efficiency
– Plaque enumeration using SalmonellaG003 strain in dual agar plate assay
– Automated plaque counting
– CL2 Biological facility
 System suitable for benchtop testing through to manufacture of kg batches of product
Conclusion
CASE STUDY: BACTERIOPHAGE ENCAPSULATION

19. 23 July 2018 P2777-P-024 v1.019Commercially confidential
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20. 23 July 2018 P2777-P-024 v1.0
An outstanding range of skills to deliver results
SKILLS AND CAPABILITIES
SENSORS
RF & ANALOGUE
ELECTRONICS
DIGITAL SIGNAL
PROCESSING
APPLICATION &
EMBEDDED SOFTWARE
USER
INTERFACES
MICRO & MACRO
FLUIDICS
ALGORITHMS
MECHANICAL
INDUSTRIAL
DESIGN
HUMAN FACTORS & USER
CENTRIC DESIGN
REGULATORY
MOLECULAR
BIOLOGY
DESIGN FOR HIGH
VOLUME MANUFACTURE
AUTOMATION AND
PROCESS DESIGN
ELECTROCHEMISTRY &
FUNCTIONALISATION
MEMS DEVICES CHEMISTRY
MARKET
RESEARCH

21. 23 July 2018 P2777-P-024 v1.0
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