This work highlights the importance of collaborations to accelerate vaccine process development and manufacturing under the constant pressure of emerging diseases and the growing need of global immunizations.
We are collaborating with the Jenner Institute of the University of Oxford to advance the development of a rapid, scalable and GMP compliant process for simian adenoviruses used as vector for vaccines such as Rabies and emerging threats like Zika and Ebola. This webinar will describe the transition from a labor and time intensive process development to one utilizing a maximum of disposable technologies such as single use bioreactors and filtration technologies, using the rabies vaccine as a first candidate. We will highlight the challenges and their corresponding solutions that in the end created a template that can be used for different types of adenoviral vectors-based vaccines manufacturing.
In this webinar, you will learn:
- The challenges of creating a rapid and scalable process for Adenovirus vector manufacturing.
- The solutions that overcame those challenges.
- How public-private collaborations can accelerate vaccine process development.
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Developing a single use adenovirus-vectored vaccine process through public-private collaboration
1. Merck KGaA
Darmstadt, Germany
Anissa Boumlic-Courtade, PhD
Associate Director EMEA Vaccines & Viral Therapies segment
Process Solutions
Life Sciences
Through a public-private collaboration
Developing a single
use adenovirus
based vaccine
process
2. Developing a single use adenovirus-based vaccine Webinar
The life science business of
Merck KGaA, Darmstadt, Germany
operates as MilliporeSigma
in the U.S. and Canada.
3. Agenda
▪ Introduction
▪ Viral vectors
▪ Jenner Institute-Oxford University project
▪ Overview of single use viral vector process
at manufacturing scale
▪ Conclusions
▪ Acknowledgement
Developing a single use adenovirus-based vaccine Webinar
4. Developing a single use adenovirus-based vaccine Webinar
Vaccines save and improve lives
And yet they’re still not accessible to everyone
• Immunization saves up to 3 million children
each year*
• But in 2015, nearly 19,4 million children
missed out on the basic vaccines they
need to stay healthy*
Shortages of certain vaccines because of
unforcasted demand and/or long & aging
manufacturing processes
Pandemics and outbreaks are
unevitable but the response is often slow
A number of emerging pathogens are still
without commercially available vaccines *source: unicef
5. Challenges & opportunities for vaccine development & manufacturing
Developing a single use adenovirus-based vaccine Webinar
Pathogen
Funding
Regulatory &
laws
Technology
Market
Manufacturing
process
Manufacturer-
supplier
One health
Collaborations
Cost
6. Different types of Vaccines
Different technologies
6
Virus
Live attenuated
virus (Influenza)
Inactivated virus
(Influenza, Polio)
Recombinant
Virus-like particle
(HPV Gardasil,
Cervarix)
Recombinant
viral vector
(Ebola)
Synthetic
nanoparticle
Bacterial
Bacterial sub-unit
(Diphteria,
Tetanos)
Glyconconjugated
polysaccharide
(Pneumococcus,
MenB, GBS)
Recombinant
protein
Nucleic acid
DNA (animal
vaccines, Inovio
Zika)
RNA (Rabies,
cancer)
Pol-
ysaccharide
Z RSV Pre-fusion F
protein
RSV Pre-fusion F
protein
7. Developing a single use adenovirus-based vaccine Webinar
New technologies: Using platforms to accelerate vaccine development
Example of Ebola 2014/15 pandemic
Company Vector type Latest Phase
MSD/New Links
Profectus Biosciences
VSV III -Filling
GSK/Okairos
J&J
Jenner Institute, Oxford
University
Vaxart
Jiangsu CDC
Adenovirus III
Bavarian Nordics
Emergent Biosolutions
MVA III
Innovio DNA II
Novavax Nanoparticle I
Thomas Jefferson
University
Rabies virus I
University
Queensland/Lyon/QIMR
VLP I
Majority of platforms based
on viral vector
Strong reduction of time
between Phase 1-3
8. Are genetically modified viruses used as
tools for antigen expression, gene delivery or
tumor targeting.
Their therapeutic applications can therefore
fall into:
• Vaccine
• Gene therapy
• Cancer treatment
Viral vectors
Developing a single use adenovirus-based vaccine Webinar
9. Global Viral vector manufacturing
High growth drivers
Emerging diseases
+20%
CAGR*
(billion USD)
Genetic disorders
Personalized
medicines
Availability of funding
Highest growths in
North America & Asia
Effectiveness of vector
based vaccines & gene
therapy
Source: Markets & Markets, 2018
Developing a single use adenovirus-based vaccine Webinar
10. Viral Vector Segmentation
Clinical trials and market share
Number of clinical trials registered on vectors globally
Vector system Number of registered trials Share in the total number
of trials (%)
Adenovirus 547 22,2
Retrovirus 478 19,4
Naked/Plasmid DNA 442 17,9
Adeno-associated Virus 204 8,3
Lentivirus 196 8
Vaccinia Virus (MVA) 175 7,1
Poxvirus 107 4,3
Herpes Simplex Virus 93 3,8
Other Vectors 223 9
Source: the Journal of Gene Medicine
11. Objectives:
Faster and cheaper template for efficient
and safe vaccine or viral therapies
This template would allow:
Accelerating clinical phases
Reducing time to market
A faster response to outbreaks and
pandemics
Meeting the need for affordable medicines
Viral vector
manufacturing
Developing a single use adenovirus-based vaccine Webinar
12. Developing a single use adenovirus-based vaccine Webinar
Viral Vector Manufacturing
Schematic viral vector process
Raw materials Media prep Amplification &
inoculation
Cell culture Clarification
(I & II)
Bioburden
removal
Nucleic acid
digestion
ConcentrationBioburden
removal
AEX ChromConcentration
& Diafiltration
FormulationSterile
filtration
Final Filling
Lysis
SEC or
AEX chrom
Upstream Purification
Formulation & Fill Finish Purification
14. Objectives
Improve the manufacturing process
for adenovirus based vaccines for
Phase 1 materials.
Develop a cost-effective and
transferable manufacturing template
that can be used to accelerate vaccine
development and manufacturing
worldwide.
Collaboration with
Oxford University
15. Objectives:
• GMP process
• Easy to operate
• Single use
• >50% efficiency
• Phase 1 scale: >5x1013 VP (1000 doses)
• Readily scalable to 5x1014
• Vaccine candidate: Rabies vaccine
« ChAdOx2-RabGP »
platform for vaccines
Depth
filtration
Developing a single use adenovirus-based vaccine Webinar
16. Advantages
1
2
Induce a broad immune
response against the target
antigen. Simian adenovirus
itself is not neutralized like
human adenoviruses
Can be engineered to be
non-replicating in the
vaccine recipient, lacking
molecular mechanism for
host genome integration
3
4
Efficiently infect a variety of
mammalian cell types
(including antigen-presenting
cells)
Amenable to scalable
manufacturing processes such as
the use of stirred tank bioreactors,
high capacity filtration methods,
and chromatographic purification
procedures
Why Simian Adenovirus Vector?
Developing a single use adenovirus-based vaccine Webinar
17. Transition to a New Manufacturing Process
Reduced handling, compressed process
Developing a single use adenovirus-based vaccine Webinar
Benzonase®
Nuclease
Clarification
Millistak+®
filters
Concentration
Pellicon® 2
cassettes
Membrane
chromatography
NatriFlo®
membrane
Diafiltration/
Formulation
Pellicon® 2
cassettes
Final filtration
Durapore® filters
Centrifugation Ultra-centrifugationShake flasks
Mobius®
Bioreactor
18. Transition to new process
Upstream
Developing a single use adenovirus-based vaccine Webinar
Mobius® 3L Bioreactor
• 2X 3L vessels
• HEK293/HEK293 T-Rex
• HEK293 chemically defined media
• Shake flask yield: 3-6 108 VP/mL
HEK293 seed
train
Cell
density at
infection
MOI Time of
harvest
Cell
density at
harvest
Yield
(qPCR)
1.5-2 x 106 3 42H 1.2 x 106
VP/mL
~5x1010
VP/mL
19. Transition to a new process
DNA reduction with Benzonase® Nuclease
Developing a single use adenovirus-based vaccine Webinar
Rationale:
- Regulatory requirements of 10ng/dose
- Impact on viscosity
- Impact on DSP performances
Adding Benzonase®
Nuclease at 60 U/mL of cell
culture decreased the level
of host cell DNA >1500 fold
during 2h lysis in bioreactor
and meet < 10ng/dose
requirement
Sample – Set point Host DNA ng/mL
Bioreactor – pre-lysis 1850
Bioreactor – post lysis 1350
Benzonase® Nuclease addition 1650
30 min 8.6
1h 2.9
1h 30 min 1.4
2h 1.1
3h 30min 0.4
Method:
- DNA reduction during in-bioreactor cell lysis
- 60 U/mL of cell culture
- Residual host DNA was assessed by qPCR
targeting Alu repeats
20. Transition to new process
Clarification using depth filtration
Developing a single use adenovirus-based vaccine Webinar
Rationale:
- Replacement of the centrifugation unit operation
- Fully disposable
- Objective: >90% virus recovery
Depth filter Name Media type Description
Millistak+® filter C0HC Double layer, High capacity
Cellulose
Diatomaceous earth
Nominal micron rating 0.2-2µ
Millistak+® HC Pro
filter
C0SP Synthetic material
Nominal micron rating 0.2-2µ
Millistak+® filter CE50 Single layer
Cellulose
Nominal micron rating 0.6-1µ
Clarisolve® filter CS20MS Polypropylene fibers
Diatomaceous earth
Nominal micron rating
Clarisolve® filter CS60HX Polypropylene fibers
Nominal micron rating
Method:
- 3 Millistak+® & 2 Clarisolve® depth
filters were tested (different grades
& charge)
21. Transition to new process
Clarification using depth filtration
Developing a single use adenovirus-based vaccine Webinar
• Millistak+® HC Pro COSP filter achieves a single-step turbidity reduction 70 ~7 NTU, with
23 cm2 device (µPod® device) allows to process > 2.3L without reaching a true Pmax
• Virus recovery: ~90% virus recovery (qPCR)
C
0
H
C
C
0
S
P
C
S
6
0
H
X
C
S
2
0
M
S
C
E
5
0
0
2 1 0 8
4 1 0 8
6 1 0 8
8 1 0 8
IU a s s a y o f F ilte r s tu d y p ro d u c ts
F ilte r ty p e
IU/ml
N o F ilte r
0 .2 2 u m filte r
22. First tangential flow filtration step
• 300 kDa Pellicon ® 2 Mini filter (0.1m2) with
Biomax® membrane
• 5L starting volume after clarification (Millistak+®
HC Pro C0SP filter + 0.2µ filtration)
• 10x volume concentration
• 10 DV filtration against IEX-suitable buffer
(containing 100 mM NaCl)
• Permeate control
Transition to new process
Purification
Developing a single use adenovirus-based vaccine Webinar
~80-88% product
recovery
20-30-fold reduction of
host cell protein
contaminants
23. Permeate flow (flux) control to a fixed, robust setpoint throughout a process is achieved through the use
of a permeate pump – the flux controlled TFF processes are also referred to as 2-pump TFF
Transition to new process
Recommendation for TFF: Permeate control
Q
Q
Permeate control allows
a stable low TMP and
ensures a better virus
recovery
24. 1 single step single use Anion exchange
chromatography using Natriflo® HD-Q
membrane
Transition to new process
Purification
Developing a single use adenovirus-based vaccine Webinar
• Quaternary amine grafted on
macroporous Hydrogel coated
PP membrane
• Membrane deliver binding
capacities that exceed resin-
based columns with fast flow
rates typical of membrane
adsorbers
• Highly versatile, and can be
utilized in flow-through or
bind-elute mode, with nearly
any ion exchange
Method
Natriflo® HD-Q Recon Mini 0.2 ml
Bind&Elute mode
Loading: ~3.30 x 1011 VP
Flow rate: 2m/min
Equilibration & wash buffer:
50 mM NaPhosphate pH 6.5, 5%
sucrose,
100 mM NaCl, 1 mM MgCl2,
0.1 % Tween®20 surfacant
Elution buffer:
50 mM tris-HCl pH 8.0, 1M NaCl, 5%
sucrose, 1 mM MgCl2, 0.1 % Tween®
20 surfacant
25. Gradient elution Natriflo® HD-Q membrane
Transition to new process
Purification
Developing a single use adenovirus-based vaccine Webinar
Natriflo® – HD Q
membrane
performance:
75-80% recovery
Good purity (SDS
Page)
26. New Adenovirus Process
Performance summary
Developing a single use adenovirus-based vaccine Webinar
Benzonase®
Nuclease
Millistak+ HC
pro C0SP
filter
Pellicon® 2
cassette
NatriFlo®
membrane
Pellicon® 2
cassette
Final filtration
Durapore ® 0.2µ
filter
Mobius®
Bioreactor
Upstream Lysis & Nucleic
acid digestion
Clarification Concentration Chromatography Concentration
& formulation
Final filtration
~5E+10
VP/mL
60 U/ml
2H
37°C
1500 fold
DNA
reduction
2.3L: 23 cm2
90%
recovery
5L: 0.1 m2
300KDa
80-90%
recovery
1 step
Bind & Elute
70-75%
recovery
0.1 m2
Ongoing
optimization
Ongoing
optimization
2 days 3 days
27. The new process at 3L scale is based on a single
use flowpath
• Bioreactor
• Tubings & liners
• Collection bags
• Filters
• Chromatography membrane
Transition to New Process
Integration of Single Use technologies
Developing a single use adenovirus-based vaccine Webinar
28. Single Use Platforms for Viral Vector
Bioreactor
ConcentrationChromatographyConcentration &
diafiltration
Clarification
Lysis & nucleic acid digestion
Final
formulation
and filtration
To filling
Media & Buffer
preparation
Up to 2000 L scale
Developing a single use adenovirus-based vaccine Webinar