Low-Energy-Electron Irradiation as a potential game ...
Transcript of Low-Energy-Electron Irradiation as a potential game ...
Low-Energy-Electron Irradiation
KyooBe Tech GmbH, Leinfelden, Germany
A game changer for inactivation of pathogens
Robert-Koch-Institute: Dashboard on Covid-19 cases / day for Germany
The Importance of Vaccines
• Vaccines have been used for over one century to eradicate some of the most severe diseases in the world
• Currently, COVID-19 ist the best evidence why vaccines have a significant position in worldwide healthcare architecture
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Bringing Inactivated Vaccine Technology to the Next Level
• KyooBe seeks to revolutionize vaccine manufacturing through advanced technology approaches
• Rapid and safe without toxic components
• Protecting important antigen structures more effectively
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Who we are
Interdisziplinary
Close to the Customer
Extraordinary
Innovative
StartUp company formed Dec. 2019 as part of Bausch+Ströbel Group:
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Our NetworkTogether for the best solutions
Part 1 - Core Technology
The Use of accelerated electrons
The technology meets the following criteria:
• LEEI is based on cathode ray technology (CRT) which has been used for decades
• Safe application in various business fields, including printing and packaging industry
• Manufacturing set up can be designed as a continuous or batch-driven process
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Applications and technologies – industrial scale
Television Ink Crosslinking Packaging / Sterilization Vaccines(starting 2023)
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The Use of acceleratedelectrons
Applications and technologies –industrial scale
Low doses(up to 1 kGy)
Medium doses(up to 10-30 kGy)
High doses(over 25 kGy)
How it works
Low-Energy Electron Irradiation (LEEI)
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Benchmarking against inactivation techniques and procedures
*Currently the only way to produce inactivated vaccinesReference: Fertey, Jasmin; Thoma, Martin; Beckmann, Jana; Bayer, Lea; Finkensieper, Julia; Reißhauer, Susann et al. (2020): Automated application of low energy electron irradiation enables inactivation of pathogen- and cell-containing liquids in biomedical research and production facilities. In Scientific reports 10 (1), p. 12786. DOI: 10.1038/s41598-020-69347-7.
Low-Energy Electron Irradiation (LEEI)
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Benchmarking against inactivation techniques and procedures
*Currently the only way to produce inactivated vaccinesReference: Fertey, Jasmin; Thoma, Martin; Beckmann, Jana; Bayer, Lea; Finkensieper, Julia; Reißhauer, Susann et al. (2020): Automated application of low energy electron irradiation enables inactivation of pathogen- and cell-containing liquids in biomedical research and production facilities. In Scientific reports 10 (1), p. 12786. DOI: 10.1038/s41598-020-69347-7.
Low-Energy Electron Irradiation (LEEI)
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Benchmarking against inactivation techniques and procedures
*Currently the only way to produce inactivated vaccinesReference: Fertey, Jasmin; Thoma, Martin; Beckmann, Jana; Bayer, Lea; Finkensieper, Julia; Reißhauer, Susann et al. (2020): Automated application of low energy electron irradiation enables inactivation of pathogen- and cell-containing liquids in biomedical research and production facilities. In Scientific reports 10 (1), p. 12786. DOI: 10.1038/s41598-020-69347-7.
Low-Energy Electron Irradiation (LEEI)
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Benchmarking against inactivation techniques and procedures
*Currently the only way to produce inactivated vaccinesReference: Fertey, Jasmin; Thoma, Martin; Beckmann, Jana; Bayer, Lea; Finkensieper, Julia; Reißhauer, Susann et al. (2020): Automated application of low energy electron irradiation enables inactivation of pathogen- and cell-containing liquids in biomedical research and production facilities. In Scientific reports 10 (1), p. 12786. DOI: 10.1038/s41598-020-69347-7.
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eBeam-based effects: molecular level
(1) Direct action
• deposition of energy by accelerated electron in the target molecule
• eliminating H atom and leading to a radical
(2) Indirect action
• Reactive species are formed in the surrounding of the target molecule
• OH radicals interact with the target resulting in strand breaks
Reference:
Hutchinson, Franklin (1985): Chemical Changes Induced in DNA by Ionizing Radiation. In Waldo E. Cohn, Kivie Moldave (Eds.): Progress in Nucleic Acid Research and Molecular Biology, vol. 32: Academic Press, pp. 115–154. Available online at http://www.sciencedirect.com/science/article/pii/S0079660308603475.
(1) e- / (2) HO
Strand Break
Nu
clei
cA
cid
s
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➢ eBeam mainly addresses large molecules like nucleic acids
➢ maintaining the antigen structure of the pathogen
eBeam-based effects: pathogen inactivation
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KyooBe‘s vision on technologyAdaptive inactivation platform for commercial manufacturing
ELLI300 (Fraunhofer IZI) Pathogen Inactivation Platform (PIP) / 2023
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KyooBe‘s vision on technologyAdaptive inactivation platform for commercial manufacturing
ELLI300 (Fraunhofer IZI) Pathogen Inactivation Platform (PIP) / 2023
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PIP will be designed as afull radiation protection systemwith biological containment!
KyooBe Tech GmbH
Ebeam accelerator
Containment box
Fluid transportcontainer
Pathogen Inactivation Platform (PIP)
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Core functions
1. Irradiation chamber
2. ebeam accelerator
3. Liquid roll-system
4. Rotating roll
5. Thin liquid film
6. Squeegee / wiper lip
7. Peristaltic pump
8. Pathogen solution
9. Inactivated solution
Containment Box #1
Containment Box #2
1.8 m
1.5 m
Key characteristics of the process
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Controlling critical process parameters
Schematic layer set-up Detailed view on liquid film environment
1 Ebeam accelerator
(Ebeams)3 Gaps - consisting of defined inert gas atmosphere
4 Gaps - separating the defined inert gas atmosphere
2 Electrons travelling through definedTi foil into aseptic space in cassette
5 Liquid pathogen film irradiated byLEEI beams – thickness up to 150 µm, depending on the process
6 Metal roll for fluid transport – also source of Bremsstrahlung
Parameter Value Impact on
Liquid film thickness ~ 120 µm throughput
Acceleration voltage 200 keV penetration depth
Ebeam current 10 mA radiation dose
Thickness Titaniumiumwindow
15 µm penetration depeth
Air gap distance 12 mm penetration depeth
Ebeam accelerator window 240 x 60 mm irradiated area
Irradiated area 160 x 60 mm throughput
Performance max. 2 kW current and voltage
Soll dose in target ~ 60 kGy pathogen inactivation
Rotational speed ~ 9 m/min throughput; radiationdose
Revolutions per minute ~ 19,1 RPM throughput; radiationdose
Throughput ~ 10 L/h Rotational speed; ebeam current
Key characteristics of the process
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Controlling critical process parameters
D = Irradiation dose [kGy]Y = Dose constant [kGy*m2*mA-1*min-1]vL = Rotational speed [m* min-1]IB = Current [mA]bB = Beam width [m]
Schematic layer set-up
Key characteristics of ebeam
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Uniformity, profile and robustness
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Do
se [
kGy]
depth [µm]
Penetration depth in material (water) at 200 keV
one Titan window
0 window (water in vacuum)
two Titan windows
Width B = 160 mm
Ebeam Accelerator WindowX = 240 mm
Eb
eam
Acc
eler
ato
r Win
do
wY
= 60
mm
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le⌀
= 15
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Do
se [
kGy]
depth [µm]
Penetration depth in material (water) at 200 keV
one Titan window
0 window (water in vacuum)
two Titan windows
Key characteristics of ebeam
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Uniformity, profile and robustness
Width B = 160 mm
Ebeam Accelerator WindowX = 240 mm
Eb
eam
Acc
eler
ato
r Win
do
wY
= 60
mm
Ro
le⌀
= 15
0 m
m
Key characteristics of ebeam
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Uniformity, profile and robustness
Width B = 160 mm
Ebeam Accelerator WindowX = 240 mm
Eb
eam
Acc
eler
ato
r Win
do
wY
= 60
mm
Ro
le⌀
= 15
0 m
m
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240 mm
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Do
se [
kGy]
depth [µm]
Penetration depth in material (water) at 200 keV
one Titan window
0 window (water in vacuum)
two Titan windows Axial length (diameter)
Longitudinal width
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Substantiating the technology
• Process Control
• Liquid film thickness (already started)
• Inline & real-time dosimetry
• Experiments and Test Cases
• Generating liquid films
• Controlling liquid film thickness
• Transporting fluids within the system
• Ebeam irradiation with Ebeam supplier (ECAB)
• Investigating continuous dosimetry
Indicator
Linear guidesystem
Micrometer screw
Electromagnetic clutch
Liquid film thickness – test case set-up ; validation by a confocal sensor(not shown here)
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In a nutshell – Status quo
*Not yet tested
• PIP = fridge-sized platform
• PIP will include an enclosed box for aseptic handling of pathogens (containment
container)
• 10 L/h* Throughput
• Within sub-seconds can pathogens be inactivated
• 60 kGy effective inactivation dose
• Ebeam accelerator for next-gen prototype platform
• Dimensions: 700 mm x 560 mm x 340 mm
• 200 kV; 10 mA; 2 kW
Early draft of role dimensions
The ebeam accelerator
Width B = 160 mm
Ebeam Accelerator WindowX = 240 mm
Eb
eam
Acc
eler
ato
r Win
do
wY
= 60
mm
Ro
le⌀
= 15
0 m
m
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Ebeam in vaccinemanufacturing
The technology meets the following criteria:
Technology Summary
Safe Applifaction
Pathogen Inactivation
ApprovedTechnology
ScalableProcess
Part 2The Application
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A disruptive change is about to happen
Cultivation process/ Harvest
Purification process
Inactivation
Formulation
Fill, Finish, Batch Release, Transport
Supply/ Testing of Raw Material ~ 2 weeks
~ 52 weeks
~ 1 week
Traditional and novel process chains
Reference: Lücke, Baedeker, Hildinger, BCG.Biotechnology report 2016
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A disruptive change is about to happen
Cultivation process/ Harvest
Purification process
Inactivation
Formulation
Fill, Finish, Batch Release, Transport
Supply/ Testing of Raw Material ~ 2 weeks
~ 52 weeks
~ 1 week
Traditional and novel process chains
Cultivation process/ Harvest
Purification process
Inactivation
Formulation
Fill, Finish, Batch Release, Transport
Supply/ Testing of Raw Material ~ 2 weeks
~ ? weeks
~ 1 week
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A disruptive change is about to happen
Cultivation process/ Harvest
Purification process
Inactivation
Formulation
Fill, Finish, Batch Release, Transport
Supply/ Testing of Raw Material ~ 2 weeks
~ 52 weeks
~ 1 week
Traditional and novel process chains
Cultivation process/ Harvest
Purification process
Inactivation
Formulation
Fill, Finish, Batch Release, Transport
Supply/ Testing of Raw Material ~ 2 weeks
~ ? weeks
~ 1 week
Cultivation process/ Harvest
Purification process
Inactivation
Formulation
Fill, Finish, Release, Transport
Supply/ Testing of Raw Material ~ 2 weeks
~ ? weeks
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Application of LEEI Technology
Product type Human Vaccines Veterinary Vaccines
Application/ Proof of concept
Pathogen inactivation Pathogen inactivation
TLR Score 8 8
Market availability 2023 2023
Basic Technology Research
Feasibility Proof-Research
Technology Development
Technology Demonstration
1-2
3-4
System/ Subsystem development
5
6-7
8
9System Test, Launch & Operations
Product type Cell- Products Blood Products
Application/ Proof of concept
Irradiation of (immune) cell therapeutics
Irradiation of transfusion-medicine products/
Serum for cell culture applications
TLR Score 5 3
Market availability To be determined To be determined
Key market
R&D / sidekicks
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Review on Feasibility
Overview of Pathogens successfully inactivated by LEEI:
Pathogen Type Veterinary / Human Concentration Dose for
inactivation
RSV ss RNA virus Human 2 x 107 TCID50/ml 20 kGy
Influenza A (H3N8) (-) ds RNA virus Veterinary 5 x 105 TCID50/ml 22 kGy
ZIKV ss RNA virus Human 5 x 106 TCID50/ml 20 kGy
PRRSV ss RNA Veterinary 5.42 log TCID50/ml 10,4 ±1 kGy
EHV-1 ds DNA Virus Veterinary 3.89 log TCID50/ml 10,4 ±1 kGy
R. pneumotropicus Bacterium Veterinary 1 x 105 CFU/ml 20 kGy
E. coli Bacterium Human 1.67 x 107 CFU/ml 2.2 kGy
B. cereus Bacterium Human 4.33 x 106 CFU/ ml 33 kGy
Eimeria tenella Parasite Veterinary 1.0- 2.0 x 105 Oozysten/ml 1 kGy
Inactivation by LEEI works for• Bacteria• Viruses• Parasites• Human & veterinary pathogens.
Dose range: 1 kGy and 33 kGy.
Inactivation is inter alia pathogen specific
0 5 10 15 20 2510 0
10 2
10 4
10 6
10 8
TC
ID5
0/m
L
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10 0
10 2
10 4
10 6
Influenzavirus H3N 8
TC
ID5
0/m
L
dose [kGy]
0 2 4 6
10 0
10 2
10 4
10 6
10 8
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Proof of Concept
Dose- Inactivationcurves
→ Successful dose- dependent inactivation of bacteria and viruses
→ Doses required for inactivation comparable to those reported with other ionizing radiation technologies.
Correlation between genome size and irradiation dose required for complete inactivation: smaller genome size -> Higher irradiation dose.
RSV
*
*
0 20 40
10 0
10 2
10 4
10 6
B. cereus
dose [kGy]
cfu
/ml
*
E.coliB. cereus
Influenza virus H3N8
dose [kGy]
dose [kGy] dose [kGy]
*
cfu
/ml
cfu
/ml
TCID
50
/ml
TCID
50
/ml
dose [kGy]
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→ High degree of conservation of the native antigen structure.
→ High reproducibility of antigen conservation.
→ Monoclonal antibody recognition of RSV F-Protein is not altered after LEEI-treatment
→ Direct comparison to FI:
Fertey, Bayer et al (2020). 10.3390/vaccines8010113.
Bayer, Fertey (2018). 10.1016/j.vaccine.2018.02.014.
B. cereus E. coli
0 2,2 6,6 negative
0
20
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% s
igna
l a
t O
D4
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/520
nm
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to p
os.
co
ntr
ol
84
0.0 2.2 11.0 16.5 22.0
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igna
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D4
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/520
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Influenza virus H3N8 RSV
% s
ign
al r
el. t
o
un
trea
ted
sam
ple
% s
ign
al r
el. t
o
un
trea
ted
sam
ple
dose [kGy]
dose [kGy]
dose [kGy]
dose [kGy]
Proof of Concept
Dose Antigenicity
0 20 25
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0 33 negative
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84
% s
ign
al r
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o
un
trea
ted
sam
ple
% s
ign
al r
el. t
o
un
trea
ted
sam
ple
85
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→ All animals developed significant levels of RSV- specific antibodies
→ All animals developed significant amounts of virus neutralizing antibodies
→ Animals immunized with 20 kGy-irradiated RSV have RSV-RNA levels close to detection limit
Binding Antibodies- RSV
Proof of Concept
Dose- ImmunizationData RSV
non
imm
unize
d
20kG
y LEE
I
25kG
y LEE
I0
5
10
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log
2neutr
alizi
ng
anti
bo
dy t
iter
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****
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imm
uniza
tion
afte
r pr
ime
afte
r bo
ost
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U
Neutralizing Antibodies- RSV
non
imm
unize
d
20 kGy
LEEI
1×10 1
1×10 2
1×10 3
1×10 4
1×10 5
1×10 6
vir
al
geno
me c
op
ies
per
45
ng
RN
A
***
Virus load in lungs
Paving the way to market
User
Product
Regulatory
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Radiation protection
GxP
GxP
Purity
Shelf life
Stability
Formulation
Scale
Demand
Production environment
Safety
Effectiveness
Cost
Procedures
Documentation
Equipment
Processes
Containment
Patient
GxP
Safety
MarketEnviron
ment
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Timeline and Milestones
2020
2021
2022
2023
2012
2014
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Partnering withKyooBe Tech
• Development (sparring) partners
• Lead customers
• Regulatory experts with vaccine manufacturing background
Enthusiastic and motivated people who want to bring WIV vaccine manufacturing to the next level