Watch the presentation of this webinar here: https://bit.ly/3lNmkf7 The therapeutic potential of mRNA has been studied for decades and this exciting modality could potentially disrupt the biological market, in particular vaccine and novel therapies. This webinar will highlight the potential of mRNA therapies and focus on the manufacturing process's associated challenges, solutions and perspectives from synthesis to delivery. mRNA has emerged as a promising modality for a wide range of therapeutics and vaccines and could become the break-through technology of this century. mRNA-based platform technologies could enable a more rapid response to infectious diseases, outbreaks or pandemics and allow efficient gene replacements or cancer treatments. mRNA represents a safer alternative to DNA-based therapies and the technology has recently advanced to overcome stability and efficacy challenges. Because of that, the industrialization of this technology is just in its infancy stages and bottlenecks exist around scalability, purity, and delivery which are key to establish and deliver the promise of such platform. This webinar will shed light on the potential of mRNA therapies and focus on the manufacturing process's associated challenges, solutions and perspectives from synthesis to delivery. In this webinar, you will learn: • The potential behind using mRNA as a therapeutic and vaccine • The mRNA production process • The challenges around mRNA production • The solutions and perspectives for a robust manufacturing process • mRNA delivery systems and their manufacturing

1. Merck KGaA
Darmstadt, Germany
November 19, 2020
A Manufacturing Perspective
Unlocking the potential
of mRNA Vaccines and
Therapeutics
Dr. Nargisse El Hajjami
Associate Director Cell & Gene Therapy Segment EMEA
Mag. Manuel Brantner
Associate Director Vaccine & Plasma Segment EMEA

2. 2 Unlocking the potential of mRNA Vaccines and Therapeutics Webinar
The life science business of
Merck KGaA, Darmstadt, Germany
operates as MilliporeSigma
in the U.S. and Canada.

3. mRNA Technology Overview
Considerations for mRNA
Manufacturing
Conclusions & Perspectives
Agenda

4. mRNA Technology
Overview

5. Delivering
the message
 Bringing an antigen either directly or using cell
host machinery to develop immunity against a
given pathogen
 Inducing a gene modification or a protein
replacement to correct a genetic defect or block
tumor progression
The purpose of vaccines and gene
mediated therapies is to trigger a
response or correct a defect
Unlocking the potential of mRNA Vaccines and Therapeutics Webinar5 Image: Courtesy of Shutterstock

6. Re-imagining vaccine manufacturing - Anissa Boumlic6
Delivering the message
Different vectors are used in the biotech industry
Viral Non - Viral
Approved and industrialized platforms
for both vaccines & gene therapy
Improved safety in the last decade
Improved safety
Simplified manufacturing process
Reduced manufacturing cost & time
Potential immunogenicity & other side
effects
Manufacturing process can be complex
High titers needed in gene therapy
formulations
Platforms still need to be established and
industrialized
Delivery to the host can be challenging
Formulations are still evolving to increase
stability and potency
Unlocking the potential of mRNA Vaccines and Therapeutics Webinar6

7. The rise of non viral technologies
Pipeline & Patents overview
Vaccines – 10% CAGR until 2025
Gene therapy – 33.1% CAGR until 2024
*Source: https://www.who.int/publications/m/item/draft-landscape-of-covid-19-
candidate-vaccines
**Nature: https://www.nature.com/articles/s41587-019-0402-x#Sec2
23% of COVID-19 vaccines in
clinical studies are using nucleic
acids technology*
>10% of Gene therapy
patents are for non viral
vehicles**
67
26
25
21
19
18
1
1
1
198
16
3
T-cell based
Non Replicating Viral Vector
VLP
Replicating Bacteria Vector
Live Attenuated Virus Non-Replicating Viral Vector
DNA
Inactivated
Replicating Viral Vector
RNA
Protein Subunit
Vaccine COVID-19 Candidates By Modality

8. The rise of non-viral technologies
Different types of nucleic acids & delivery mechanisms
8
Naked Encapsulated
DNA RNA
Target gene
DNA
Plasmid DNA is the
common approach
mRNA
Lipid nanoparticles
formation using lipids
and/or polymers to
protect from nucleases &
endosomes
Intravenous or lymphatic
injection route
siRNA Antisense
RNA
Local administration
at target site
Rapid degradation
Intratumoral,
intranodal or subq
Unlocking the potential of mRNA Vaccines and Therapeutics Webinar

9. Re-imagining vaccine manufacturing - Anissa Boumlic9
The History of mRNA
mRNA potential has been explored for more than 60 years!
1969 2015
In vitro
translation
of isolated
mRNA1
Cationic lipid-
mediated
delivery of
mRNA to cells
in vivo2
mRNA elicited
an antigen-
specific immune
response in
mice.3
1st report of
an anti-
tumor T cell
response
after mRNA
injection in
vivo4
Discovery that
nucleoside-
modified RNA
is non
immunogenic5 1st clinical trials
based on the
direct
application of
mRNA.6
Dev of TALEN
mRNA for
gene editing7
Protein
replacement with
LNPs carrying
factor IX mRNA
restores normal
protein levels in a
hemophilia B
animal model8
COVID-19
Pandemic
1961 2020
1989
19901978
1993
1994 201920171997
1999 2005
20092007
2008 2011
2013
1995
mRNA
Discovery
mRNA
delivered using
liposomes9
1st description
of IVT mRNA
translation in
vivo.10
Introduction of
self-amplifying
mRNA as a NA
vaccine4
FDA approves
first clinical trials
with ex vico DCs
loaded with
mRNA for cancer
to11
IVT mRNA with
modified
nucleotides
improves RNA
stability &
translation5
1st report on
cellular uptake of
mRNA after skin
delivery12
1st human cancer
immunotherapy by
direct mRNA
injection13
Phase I/II trials
with injection of
naked mRNA or
protamine mRNA
cancer vaccines6, 14
Development
of CRISPR-
Cas9 mRNA for
gene editing15
Personalized neo-
epitope mRNA
vaccine tested in
patients with
melanoma16
First reports of
mRNA–LNP
-formulation for in
vivo vaccines
against the Zika
virus17, 18
Clinical trials with
distinct RNA
vaccines for
infections & cancer
Trends towards
• LNP-based
delivery
Sources
1:Lockard et al 1969; 2:Malone et al 1989; 3:Martinon et al 1993; 4:Zhou et a 1994. 5: Kariko et al 2005. 6:Weide et al 2008; 7:Wood et al 2011; 8:Li B. et al 2015; 9:Dimitriadis et al 1978; 10:Wolff
et al 1990. 11:Heiser et al 1997. 12:Probst et al 2007; 13:Yoon et al 2009; 14:Rittinh et al 2011; 15:Hwang et al 2013; 16:Sahin et al 2017; 17:Pardi et al 2017; 18:Richner et al 2017

10. mRNA technology
Translated proteins replace missing proteins or induce immune responses
10
mRNA is an essential
component of the central
dogma of life
• mRNAs encodes for essential
proteins and their potential can be
used for therapeutic or
prophylactic applications
• Can encode an antigen, be a
protein replacement for a
defective gene or activate
responses against tumors
(personalized medicine)
• Rapid & transient protein
production
• No risk of insertion or mutagenesis
Unlocking the potential of mRNA Vaccines and Therapeutics Webinar

11. mRNA technology
Two types of mRNA constructs are being actively used
11
Non-Replicating mRNA (NRM) Self Amplifying mRNA (SAM)
Number of
nucleotides
2000 – 3000 nt ~ 10 000 nt
Type Single-stranded Single-stranded
Size 660 – 990 kDa 3300 kDa
Potency Low levels of proteins
High levels of proteins
Enhanced protein expression
Immunity
No theoretical risk of anti-vector immunity
with non-viral delivery systems
No anti-vector effect has been observed
yet
Potential interactions between encoded
non-structural proteins and host factors
require additional investigation.
Concentration
needed/dose
~50-200ug/dose ~1ug/dose
5’ UTR GOI 3’ UTR5’ cap A A A A A A A A 5’ UTR GOI 3’ UTR5’ cap A A A A A A A AReplicase
Unlocking the potential of mRNA Vaccines and Therapeutics Webinar

12. Market landscape
mRNA pipeline for vaccines & therapeutics
12
Data as of July 1 2020 | Source: Pharmaprojects, Citeline, EvaluatePharma updated for COVID-19
1: 1. Includes Blood, Immunology, Nephrology, Respiratory, Musculoskeletal, Ophthalmological | Data as of July 1 2020 |
Source: Pharmaprojects, Citeline, EvaluatePharma
34%
43%
31%
28%
43%
24%
9%
10%
20%
25%
4%
Total
3%
3%
3%
Clinical
4%
4% 0%4%
100%
3%
3%
4%
Pre-clinical
34138 104
Cardiovascualar
Oncology
Other
Respiratory
Hepatology
Infectious Disease / Vaccine
Neurology
Pipeline Split Across Therapeutic Area
Novel indications
expected to enter
clinic
1
22
11
11
9
9
6
40
7
6
4
3 9
9
0
15
17
29
0
Other
9
By Company (138 Assets, 34 in clinic)
23 Companies
44%
14
104
18
2
0
138
mRNA
# of Assets Preclinical Phase I Phase II Phase III/Filed Launched Total
Assets in
Clinical22%
Includes 25 COVID-
19 Vaccine
Candidates
Unlocking the potential of mRNA Vaccines and Therapeutics Webinar

13. Market landscape
mRNA technology is emerging amid COVID-19 vaccine race
13
Company
mRNA Vaccine Type
& Delivery system
Clinical
Phase
nrRNA & LNP
III
III
II
I
saRNA & LNP
I/II
I
Source: https://www.who.int/publications/m/item/draft-landscape-of-covid-19-candidate-
vaccines, as of November 12th, 2020
13% of COVID-19
vaccine in clinical
trials are based on
mRNA
12% of COVID-19
vaccines in
preclinical studies
are based on mRNA
Clinical (48 Candidates)
Preclinical (164 Candidates)
Unlocking the potential of mRNA Vaccines and Therapeutics Webinar
11%
15%
19%
30%
9%
13%
DNA
Inactivated
RNA
Non-Replicating VV
Protein Subunit
Replicating VV
VLP
4%
8%
8%
12%
35%
11%
12%
10%
1%
Live Attenuated Virus
DNA
RNA
Inactivated
Replicating VV
Non-Replicating VV
Replicating Bacteria Vector
Protein Subunit
T-cell based
VLP
2%
1%

14. Re-imagining vaccine manufacturing - Anissa Boumlic14
mRNA technology
Advantages
1 RNA therapeutics are safer
than DNA therapeutics
(RNA does not integrate
into the Genom)
RNA is not infectious
RNA is produced using a
cell-free enzymatic
transcription reaction or
chemical synthesis
2 Production of RNA-
based vaccines is
faster compared to
production of
traditional vaccines
Good scalability
3
Producing RNA
vaccines is less
expensive than
producing the full
antigen protein
4
For any outbreak
RNA vaccines are
more flexibel, any
desired RNA for any
desired protein of
interest can be
prepared in short time
for each individual
patient (personalized
medicine)
Safety
Time
COst
Flexibility
Unlocking the potential of mRNA Vaccines and Therapeutics Webinar14

15. Re-imagining vaccine manufacturing - Anissa Boumlic15
mRNA technology
Challenges
1 Single stranded
Highly negatively charged
Rapid degradation of RNA
caused by endonucleases
Cold-chain
2 Exogenous mRNA is
immunostimulatory, as it is
recognized by a variety of
innate immune receptors
In applications such as
protein-replacement
therapies, activation of the
innate immune system by
toll-like receptors by IVT
mRNA is not desired
3 The in vivo Delivery of
RNA is very inefficient
RNA vaccines have a
lower immunogenicity
compared to traditional
vaccines, so higher doses
are needed
RNA instability
Immune modulation
Efficiency
Unlocking the potential of mRNA Vaccines and Therapeutics Webinar15

16. Considerations for
mRNA Manufacturing

17. The first step is to generate a pDNA coding for the RNA polymerase promoter and the targeted
mRNA construct:
mRNA manufacturing
A plasmid DNA template is required for in vitro transcription
17
Fermentation Clarification Purification
with Chrom
(2-3 steps)
Final
Filtration
Storage
Concentration
and Diafiltration
(UF/DF)
Thaw cells
E.coli+pDNA
Cell Harvest Cell Lysis Concentration
and Diafiltration
(UF/DF)
RNA-
polymerase
pDNA
mRNA
pDNA
Unlocking the potential of mRNA Vaccines and Therapeutics Webinar

18. mRNA manufacturing
Several attractive features
18
Make Purify Formulate
pDNA
Linearization
Chromatography
And/or UF/DF
In vitro
Transcription
Chromatography
And/or UF/DF
Enzymatic
capping
Chromatography UF/DF Encapsulation
& Formulation
Final Sterile
Filtration
Same facility/process for multiple
targets
Short production time
Cell free enzymatic manufacturing
process
Simple manufacturing process
steps
Easy & rapid to develop
Unlocking the potential of mRNA Vaccines and Therapeutics Webinar

19. mRNA manufacturing
Process objectives
19
Make Purify Formulate
pDNA
Linearization
Chromatography
And/or UF/DF
In vitro
Transcription
Chromatography
And/or UF/DF
Enzymatic
capping
Chromatography UF/DF Encapsulation
& Formulation
Final Sterile
Filtration
Unlocking the potential of mRNA Vaccines and Therapeutics Webinar
mRNA
product
• mRNA Size/MW
• mRNA integrity, potency
• Encapsulation efficiency
• Capping efficiency
• Impurities: dsRNA, DNA template, nucleoside
triphosphates, RNA polymerase
• Appearance, pH, osmolality, subvisible particles,
elemental impurities and residual solvents
Quality ControlQuality Attributes
mRNA structure:
Purity: Impurities removal reduces innate sensing
promoting expression.
• 5’ Cap: Affect innate sensing and protein
• UTR’s: Maximize gene expression
• CDS (Coding Sequence): Gene expression
• 3’ Poly-A-tail: Length affects translation &
mRNA protection
Efficient delivery system

20. mRNA manufacturing
Plasmid linearization eliminates risk of transcriptional read-through
20
Make Purify Formulate
pDNA
Linearization
Chromatography
And/or UF/DF
In vitro
transcription
Chromatograp
hy
And/or UF/DF
Enzymatic
capping
Chromatography UF/DF Encapsulation
& Formulation
Final Sterile
Filtration
• The linearization reaction mix includes:
- pDNA
- WFI
- Reaction buffer
- Restriction enzyme
Incubation at 37°C for 4 hours
• To stop the reaction:
• EDTA
• Heat inactivation at 65°C
optional
Unlocking the potential of mRNA Vaccines and Therapeutics Webinar

21. mRNA manufacturing
Purification of linearized pDNA template prior transcription
21
Make Purify Formulate
pDNA
linearization
Chromatography
And/or UF/DF
In vitro
transcription
Chromatography
And/or UF/DF
Enzymatic
capping
Chromatography
UF/DF Encapsulation
& Formulation
Final Sterile
Filtration
Chromatography
Tangential Flow
Filtration
• Ion Exchange
• Size Exclusion
Impurities removal: endotoxins, DNA fragments, restriction enzymes, BSA..
Typical MWCO: 30 – 300kDa
Potentially higher impurity removal
Extra operation needed: TFF for media exchange; expensive
Linearized pDNA can be purified, concentrated and diafiltered
within the same unit operation
Efficiency of impurities removal depends on MWCO Vs pDNA size
Unlocking the potential of mRNA Vaccines and Therapeutics Webinar

22. mRNA manufacturing
In vitro transcription has two options for capping
22
Make Purify Formulate
pDNA
linearization
Chromatography
And/or UF/DF
In vitro
transcription
Chromatography
And/or UF/DF
Enzymatic
Capping
Chromatography UF/DF Encapsulation
& Formulation
Final Sterile
Filtration
• Linearized pDNA
• RNA Polymerase T7, T3 or SP6
• NTPs
• Transcription Buffer
• RNase inhibitor
• Others (Mg2+,
Pyrophosphatase.. )
Co-transcriptional
Uses 4:1 ratio
Cap-analog: GTP
Capping during transcription; Cheaper
80% efficiency; Lower yield; Non-capped impurities
Incubation at 37°C for 4 hours
DNA Digestion: DNAaseI
Capping
Option 2Option 1
Or
Vaccinia virus capping enzyme
Performed at 37°C for 1 hour Expensive; Extra unit operation
High capping efficiency (>99%)
Unlocking the potential of mRNA Vaccines and Therapeutics Webinar

23. mRNA manufacturing
Primary purification step
23
Make Purify Formulate
pDNA
linearization
Chromatography
And/or UF/DF
In vitro
transcription
Chromatography
And/or UF/DF
Enzymatic
capping
Chromatography UF/DF Encapsulation
& Formulation
Final Sterile
Filtration
• Ion-exchange
• Size-exclusion
• Affinity: Poly(dT) capture
Impurities removal: DNA fragments, endotoxins, enzymes..
Extra unit operation required: TFF performed afterwards for medium exchange,
more expensive
Efficient DNA template removal (avoiding hybridization risk)
Chromatography
Precipitation Not scalable & inadequate for commercial therapeutic use
Depth filtration Bind/elute mode Not commonly used
Unlocking the potential of mRNA Vaccines and Therapeutics Webinar
Typical MWCO: 30 – 300kDa
Watch-out: DNA fragments hybridization risk
mRNA can be purified concentrated and diafiltered within the same unit
operation
Tangential
Flow Filtration

24. Use aqueous solutions (salt gradient)
DBC ± 5 mg RNA/mL
RNA-DNA hybrids, dsRNA, hairpin contaminants
Typically followed by a second chromatography
step for polishing (IEX/HIC)
Binds specifically Poly(A) tail (full length
transcripts)
Removes: DNA, Nucleotides, Enzymes, Buffer..
mRNA manufacturing
Chromatography is an efficient purification step
24
Make Purify Formulate
pDNA
linearization
Chromatography
And/or UF/DF
In vitro
transcription
Chromatography
And/or UF/DF
Enzymatic
capping
Chromatography
UF/DF Encapsulation
& Formulation
Final Sterile
Filtration
Reversed-Phase Ion-Pair
Use aqueous solutions
DBC >10 mg RNA/mL
Elution can require chaotropic agents
and/or elevated temperature
Removes dsRNA, uncapped RNA,
secondary RNA structures (hairpin)
Ion-pair reagents form complexes
with RNA → extensive diafiltration
needed
DBC <10 mg RNA/mL
High & rapid RNA purity
Use solvents
Not ideal for scaling-up
Affinity Chromatography
Poly(dT) Capture
Unlocking the potential of mRNA Vaccines and Therapeutics Webinar
Anion-Exchange

25. mRNA manufacturing
Final concentration and diafiltration
25
Make Purify Formulate
pDNA
linearization
Chromatography
And/or UF/DF
In vitro
transcription
Chromatography
And/or UF/DF
Enzymatic
capping
Chromatography UF/DF Encapsulation
& Formulation
Final Sterile
Filtration
Achieve final concentration and exchange into final buffer
Typical MWCO: 30 – 300kDa
mRNA can be further purified, concentrated and diafiltered within
the same unit operation
Efficiency of impurities removal depends on MWCO Vs mRNA and
impurities size
Maximize product purity
Unlocking the potential of mRNA Vaccines and Therapeutics Webinar

26. mRNA manufacturing
Encapsulation is crucial for mRNA stability and delivery
efficiency
Make Purify Formulate
pDNA
linearization
Chromatography
And/or UF/DF
In vitro
transcription
Chromatography
And/or UF/DF
Enzymatic
capping
Chromatography UF/DF Encapsulation
& Formulation
Final Sterile
Filtration
Most used:
Lipids Lipid Nanoparticle
(LNP)
Polymers
Liposomes
Lipoplexes
Polyplexes
Polymer
+
LipoplexLipid
PolyplexmRNA
+
mRNA
Increased transfection rate
Allows intravenous injection
Enables active targeting
Additional safety concerns
Storage & cold chain
LNP ~200nm are challenging
Unlocking the potential of mRNA Vaccines and Therapeutics Webinar
LNP + mRNA

27. mRNA manufacturing
Scale-up considerations
27
Lab scale
• solvent extraction
• precipitation steps
• use of hazardous solvents
• scalability issues
• PD expertise
• Extraction & precipitation -> chrom and/or TFF
• GMP compliance
• RNase free/risk assessment for product contact
equipment, raw material & solutions
• Efficient & safe delivery systems
• Sterile filtration of large mRNA complexes
• Cold chain & distribution to point of care
Manufacturing Scale
Unlocking the potential of mRNA Vaccines and Therapeutics Webinar

28. Conclusions & perspectives

29. 29
In Conclusion…
Key takeaways Perspectives
The emergence of mRNA-based vaccines is boosting the
growth in other areas, such as GT and plasmid
manufacturing
mRNA vaccines may be revolutionary and become the
best solution for treating outbreaks
mRNA manufacturing paradigm need to shift to
establish a simple & robust platform at industrial scale
The mRNA constructs and delivery systems still
need to be carefully evaluated for safety,
efficacy, quality and manufacturability
Advanced technologies & novel
processing concepts:
Single use & closed processing
Next generation manufacturing in
response to “the need for speed”:
Modular & pre-engineered
solutions
Next generation of mRNA
• Sequence optimization
• Novel Delivery Systems
(Hybrid NPs, ligands-
based..)
1
2
4
1
2
3
3
Unlocking the potential of mRNA Vaccines and Therapeutics Webinar

30. We do it with you - Product & Service Portfolio
mRNA production
Oligos*
Reagents*
Mixers & tanks
Bags
Sampling
solutions
Product
characterization
Validation
services
Buffers
Benzonase®
endonuclease
TFF cassettes
and capsules
Single-use
systems &
multi-use skids
Mixers
Pleated filters
Single-use
systems
Validation
services
Clean-in-place
solutions
Solvents & Buffers
IEX resins &
Membrane-based
chromatography
Single-use
systems & multi-
use skids
Mixers
Storage
assemblies
Biosafety testing
Validation
services
Clean-in-place
solutions
Buffers
TFF cassettes
and capsules
Single-use
systems & multi-
use skids
Mixers
Storage
assemblies
Validation
services
Mixers
Bags
Sterilizing filters
SU assemblies
Sterile
connectors
Sampling
solutions
Excipients
Lipids
Buffers
Mixers
Storage
assemblies
Sampling
solutions
Biosafety
testing
Validation
services
Sterilizing filters
Integrity testers
Single-use final
fill assemblies
Sterile
connectors
Storage
assemblies
Sampling
solutions
Biosafety testing
Validation
services
30
 *supplied from Research & Applied division
Assure
mRNA
transcription
Plasmid DNA
removal
Chromatography Tangential flow
Filtration
Encapsulation
& Formulation
Final FillpDNA
linearization
Linirized pDNA
purification
Enzymatic
capping
Reagents*
Filters
Mixers
Hold bags
Sampling
solutions
Single-use
assemblies
Sterile
connectors
Product
characterization
Clean-in-place
solutions
Solvents &
Buffers
IEX resins
& membranes
Chrom
SU systems &
multi-use skids
Mixers
Biosafety testing
Validation
services
Unlocking the potential of mRNA Vaccines and Therapeutics Webinar

31. Associate Director Cell & Gene Therapy
Segment EMEA
BioProcessing, Process Solutions
Nargisse.el-Hajjami@merckgroup.com
DR. NARGISSE EL HAJJAMI Laurens Vergauwen
Process Development Scientist, EMEA, Technology
Management, Process Solutions
Patryk Kelley
Associate Segment Manager, Vaccine & Plasma Segments,
Bioprocessing, Process Solutions,
Dr Anissa Boumlic
Head of Global Vaccine & Plasma Segments
Bioprocessing, Process Solutions
Special Thanks to:
Associate Director Vaccine & Plasma
Segment EMEA
BioProcessing, Process Solutions
Manuel.brantner@merckgroup.com
Mag. Manuel Brantner

32. Millipore, SAFC, Bioreliance and the vibrant M are trademarks of Merck KGaA, Darmstadt, Germany or its affiliates. All other trademarks are the property of
their respective owners. Detailed information on trademarks is available via publicly accessible resources.
© 2020 Merck KGaA, Darmstadt, Germany and/or its affiliates. All Rights Reserved.
For more information about vaccine production, please visit:
https://www.sigmaaldrich.com/process-solutions/vaccines.html
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