In this webinar, we will discuss: • The application of RNA therapeutics and the different drug delivery routes used in the clinic. • Design principles for developing lipids-based RNA formulations. • Critical parameters to consider for cost effective development and consistent performance of RNA therapeutics and vaccines. RNA therapeutics are changing the way we address diseases. Applications range from gene therapy, oncology, to vaccines for infectious diseases such as COVID-19. The performance of RNA therapeutics critically depends on its formulation. Key decisions have to be made early on in the drug development process; choosing the appropriate drug delivery method and novel excipients. Raw material source and judicious choice of chemistry, ultimately determine the quality of novel lipid excipients which, in turn, has a big impact on the performance, reproducibility, costs, and regulatory approval timelines. This webinar will propose solutions to maximize the probability of success while formulating RNA therapeutics and vaccines. Participate in the interactive webinar now: https://bit.ly/2xXMZlm Explore our webinar library: www.merckmillipore.com/webinars

1. The life science business of Merck KGaA,
Darmstadt, Germany operates as
MilliporeSigma in the U.S. and Canada.
Key to successful
formulation development
for lipid based RNA delivery
and vaccines
Webinar 2020
Shiksha Mantri

2. The life science business of
Merck KGaA, Darmstadt, Germany
operates as MilliporeSigma
in the U.S. and Canada.
Key to successful lipid-based RNA formulations | 09.04.20202

3. Agenda
1. Introduction
Benefits and considerations for RNA therapeutics
2. Critical parameters defining activity of lipid-based formulations
Composition, source, quality, formulation process
3. Essential considerations for successful drug development
Timing, regulatory aspects, number of sources, supplier choice
4. Summary and Q&A

4. Agenda
1. Introduction
Benefits and considerations for RNA therapeutics
2. Critical parameters defining activity of lipid-based formulations
Composition, source, quality, formulation process
3. Essential considerations for successful drug development
Timing, regulatory aspects, number of sources, supplier choice
4. Summary and Q&A

5. Central dogma of molecular biology
RNA as API
Cell
Nucleus
Cytoplasm
How can RNA be a drug?
• DNA/RNA/ protein – all can be APIs
• RNA has several advantages over DNA
or proteins as API
• Rapid and transient protein
production
• No risk of insertional mutagenesis
• Cytoplasmic activity
RNAi
DNA
mRNA
Protein
Successful development for lipid based RNA delivery and vaccines, Shiksha Mantri, 2020 Image from Wikicommons5

6. Antigen/ protein production
Gene silencing/ activation
Enzyme replacement therapy
Therapeutic antibodies
Gene editing: CRISPR/Cas tech
RNAs as API expand the range of druggable targets
RNA as API – Introduction
Vaccines
Cancer
Pulmonary
Liver metabolic disorders
Precision medicine (various indications)
 Small RNAs: siRNA,
shRNA, saRNA, ASO,
etc.
 Long RNAs: mRNA
RNA as an API can be used for a variety of applications and indications
Abbreviations used:
siRNA - short interfering RNA; shRNA - small hairpin
RNA; saRNA - short activating RNA; ASO - antisense
oligonucleotides; mRNA - messenger RNA
Successful development for lipid based RNA delivery and vaccines, Shiksha Mantri, 20206

7. Example: COVID-19
RNA therapeutics
1
2
3
Antigen delivery
mRNA
Halt viral replication
 RNAi
Antibody
mRNA
Successful development for lipid based RNA delivery and vaccines, Shiksha Mantri, 2020
mRNA of
antigen
mRNA(s) of
antibody
Pathogen mRNA
7

8. When can RNA Tx and vaccines work?
Encapsulation
techniques
Chemical
modifications
ASO
siRNA mRNA
sgRNA
Abbreviations used: siRNA - short interfering RNA; sgRNA – Single
guide RNA; ASO - antisense oligonucleotides; mRNA - messenger RNA
Kowalski, et al. (2017) Genome Medicine 9(1): 60
Successful development for lipid based RNA delivery and vaccines, Shiksha Mantri, 2020
Known gene
target
RNA should
reach site of
activity
Two main
strategies
 Stability
 Immunogenecity
 Charge
 Size
 Endosomal escape
8

9. Applicability
Comparison between different methods for RNA delivery
Successful development for lipid based RNA delivery and vaccines, Shiksha Mantri, 2020
Chemical modifications Encapsulation
Vs.
 Naked (chemical modifications to
backbone, nucleotides)
 GalNac (chemical conjugation to
targeting moeity such as GalNac)
 Lipids
 Polymers, inorganic NPs, hybrid formulations
 Viral
 Others
Abbreviations used: GalNac: N-Acetyl-D-galactosamine, ERT: Enzyme replacement therapy
 Short RNAs, i.e. ASO, miRNA, siRNA  Applicable to all, including mRNA
 Naked: broad, by local delivery
 GalNac: limited, hepatocyte targeting
 Very broad incl. CRISPR, vaccines,
ERT, etc.
API
Delivery
method
9

10. Encapsulation via electrostatic interactions
Choices of delivery vehicles
Lipids
Lipoplexes
Lipid NPs
Most advanced
Granot, Y. and D. Peer (2017). Seminars in Immunology 34: 68-77.
Active RNA pipeline,
by delivery method
35
113
(30.0%)
47 81
7
94
Lipids
Naked
Galnac
Polymer
Viral Vector
Other (e.g. Gold NP)
Pre-clinical 78
Ph I 25
Ph II 7
Ph III 2
Commercial 1
Liposomes
Sources: Clinical trials: Clinicaltrials.gov, preclinical: Pharmacircle
Liposome Lipid nanoparticle (LNP)
Successful development for lipid based RNA delivery and vaccines, Shiksha Mantri, 2020
 Versatile drug delivery system
 Co-delivery of multiple RNAs, small molecule drugs
 Plug & play
 Immunostimulation
Lipoplex
10

11. Agenda
1. Introduction
Benefits and considerations for RNA therapeutics
2. Critical parameters defining activity of lipid-based formulations
Composition, source, quality, formulation process
3. Essential considerations for successful drug development
Timing, regulatory aspects, number of sources, supplier choice
4. Summary and Q&A

12. Planning transition from Lab → GMP → full scale
Typical formulation process
Successful development for lipid based RNA delivery and vaccines, Shiksha Mantri, 2020
GMPLab conditions
RNA
aqueous
Lipids
Organic
solvent
LNPs
Pre-bulk
LNPs
Buffer exchange/
dialfiltration/
sterlization
+
Formulation
process
Full scale= =
12

13. Key parameters that affect activity of the final formulation
Composition of
delivery vehicle
Quality of
components
Synthesis route Formulation
process
Performance
Performance = Activity, Stability, Success
1 3
2 4
Successful development for lipid based RNA delivery and vaccines, Shiksha Mantri, 202013

14. Ratio and type of lipids is critical
1. Composition of delivery vehicle
• Encapsulation
• Release
• Influences toxicity
• Structure
• Fusogenicity
• Reduces toxicity
• Stability
• Cargo release
• Immunogenicity
• Structural integrity
• Which Cationic/ Ionizable lipid: Structure defines potency,
targetability, immune response
• Which anionic/ neutral lipid?
• Which PEG lipid: Optimal lengths of C-chain and PEG required
• Animal-derived or Synthetic Cholesterol?
Lipid nanoparticles
Successful development for lipid based RNA delivery and vaccines, Shiksha Mantri, 2020
Cationic/
ionizable lipid
1
Anionic/neutral
lipid
2
PEG lipid
3
Cholesterol
4
Parameters to decide:
✓ Which lipids?
✓ Lipids ratios
✓ N/P ratio
✓ Administration route
14

15. Features to keep in mind while designing new ionizable lipids
Ionizable lipids design
Successful development for lipid based RNA delivery and vaccines, Shiksha Mantri, 2020
Ramishetti et al. Adv. Mater.2020, Heyes et al. J Control Release. 2005, Hassett et al. Molecular Therapy: Nucleic Acids 2019,
Rietwyk et al. ACS Nano 2017
Consider:
• Number and position of
double bonds in lipid chains
• Branched vs. Linear
• Ester groups within the lipid
chain
Consider:
• Cleavable groups for
degradability such as
esters, amides
Consider:
• Number of amine groups, type
• Shape – cyclic/ linear
• Substitution pattern of the
amine groupLinker
• In vivo efficacy
• Biodistribution
Lipid chain
• Phase transition temp.
• Fusogenicity
• Biodegradability
Head group
• pKa, shape
• Transfection efficiency
• Biodistribution/ Targetability
• Immune system activation
Dlin-MC3-DMA
15
API
Application
Administration
route
Target cell
type

16. 2. Synthesis route/ Process for a novel lipid structure
Points to consider for manufacturing a new lipid:
• How facile is the chemical synthesis? Yield?
• What is the final purity? Are isomers being produced?
• Is the reaction scheme consistently reproducible?
• Is the process scalable?
• Which steps should be done in a GMP environment?
• Detect and identify
impurities: HPLC-CAD
• Endotoxins/ Bioburden
• Solubility tests
Successful development for lipid based RNA delivery and vaccines, Shiksha Mantri, 2020
Cost =
 Number of chemical steps
 Overall yield
 Cost of starting materials
 Scale of reaction
Analy-
tics
=
16

17. Final GMP process needs to be scalable and reproducible
2. Synthesis route/ Process
1
Reduce number of synthesis steps
Define GMP steps
2
Avoid reaction conditions that lead to isomerization
Yield and quality should be reproducible
3
Consider scalability, economy of scale, batch size
early on
We
recommend
Successful development for lipid based RNA delivery and vaccines, Shiksha Mantri, 202017

18. Quality
Convenience, reproducibility, stability, release profile of formulation
3. Quality of lipids
• Crystallinity
• Solubility
• Stability
• Flowability
Consistency
• Lipid & DP
stability
• Expected
drug release
profile
• Reproducible
results
• Avoiding
bridging
toxicity
studies
Good material
characteristics
High
purity
 Expected results
 No regulatory hurdles
 Cost-saving
 Ease of drug product manufacturing
Successful development for lipid based RNA delivery and vaccines, Shiksha Mantri, 2020
implies
means
18

19. Process development → quality
Examples
DOPE
Successful development for lipid based RNA delivery and vaccines, Shiksha Mantri, 2020
• Free flowing powder
• Fast and complete dissolution
• Lumps, gel & foam
• Limited solubility even after lyophilization
Powder DOPEWax-like DOPE
DOPC
• Enhanced stability: >7 years at 25°C / 60% rH
• Fast dissoluton rate
• Easy handling
Crystalline DOPC*Amorphous DOPC
5 mW
19

20. Consistency required in every step of process
How to achieve consistent quality?
1
2
• Low level of by-products
• Defined stereochemistry (cis/trans)
• Low bioburden and endotoxin levels
• Plant-derived raw materials with BSE/TSE and non-GMO
certificates
• Use class II and III solvents
Appropriate manufacturing process
High and consistent quality raw
materials
• Manufacturing under GMP environment, ICH Q7 guidelines
• Optimal reaction conditions
• Defined batch sizes
3Good purification process
• Purification steps must be scalable as well
• Use crystallization if possible
• Employ liquid/liquid extraction methods
• Avoid chromatography
• Convert chromatography into filtration over silica gel
Successful development for lipid based RNA delivery and vaccines, Shiksha Mantri, 202020
Picture of one of our kilolabs in Switzerland

21. Key parameters that affect activity of the final formulation
Composition of
delivery vehicle
Quality of
components
Synthesis route Formulation
process
Performance
1 3
2 4
Performance = Activity, Stability, Success
Successful development for lipid based RNA delivery and vaccines, Shiksha Mantri, 202021

22. Formulation process should be reproducible and scalable
4. Formulation process
Successful development for lipid based RNA delivery and vaccines, Shiksha Mantri, 2020
Characteristics, Stability, PerformanceProcess conditions
RNA
aqueous
Lipids
Organic
solvent
LNPs
Pre-bulk
LNPs
bulk
Buffer exchange/
dialfiltration
LNPs
Final DP
Lyophilization/
fill & finish
+
Formulation
process
DP= drug product22

23. Points to consider
Role of formulation conditions
Successful development for lipid based RNA delivery and vaccines, Shiksha Mantri, 2020
Characteristics, Stability, PerformanceProcess conditions
Formulation
process
RNA
aqueous
Lipids
Organic
solvent
LNPs
Pre-bulk
LNPs
bulk
Buffer exchange/
dialfiltration
LNPs
Final DP
Lyophilization/
fill & finish
+
DP= drug product
Formulation process
• Formulation technique
• Thin film hydration
• Detergent removal
• High pressure homogenization
• Solvent injection
23

24. Formulation techniques
Recap
Successful development for lipid based RNA delivery and vaccines, Shiksha Mantri, 2020
Lipids in organic solvent
Drying step Hydration
Addition of water
(and hydrophilic drug/ antigen)
Stirring
Sonication
Extrusion
Homogenization
Multilamellar vesicles Unilamellar vesicles
Lipid film / cake
+ Purification
Thin film hydration
Solvent injection
Lipids in
organic
solvent
RNA in
aqueous
solution
Mixing
P P
P = pump
Dia/ultrafiltration
Mixing
Cross-flow mixing (Polymun)
Microfluidics mixing
24

25. Effect of formulation technique
Successful development for lipid based RNA delivery and vaccines, Shiksha Mantri, 2020
Ethanol injection
EtOH injection
+Extrusion
Cross-flow
(Polymun)
Homogenization
Dry film
hydration+
homogenization
Formulation technique Results
Data from Dr. Finn Bauer, Dr. Michael Plastcher, with Polymun, Wagner et al., J Liposome Res 2006 16(3):311-9
Formulation technique effects:
• Size, PDI, encapsulation efficiency
• Costs
• Speed
• Reproducibility
• Scalability
• Stability (for e.g. high pressure
homogenization →Lipids
oxidation, hydrolysis)
25

26. Points to consider
Role of formulation conditions
Successful development for lipid based RNA delivery and vaccines, Shiksha Mantri, 2020
Characteristics, Stability, PerformanceProcess conditions
Formulation
process
RNA
aqueous,
low pH
Lipids
Organic
solvent
LNPs
Pre-bulk
LNPs
bulk
Buffer exchange/
dialfiltration
LNPs
Final DP
Lyophilization/
fill & finish
+
DP= drug product
Formulation process
• Flow rate, injection
hole diameter
• Mixing speed
• Process temperature
Lipid concentration
• Size, PDI
N/P ratio
Solvent injection
26

27. Points to consider
Role of formulation conditions
Successful development for lipid based RNA delivery and vaccines, Shiksha Mantri, 2020
Characteristics, Stability, PerformanceProcess conditions
RNA
aqueous,
low pH
Lipids
Organic
solvent
LNPs
Pre-bulk
LNPs
bulk
Buffer exchange/
dialfiltration
LNPs
Final DP
Lyophilization/
fill & finish
+
Formulation
process
DP= drug product
Duration,
Temperature
pH
• Lipid hydrolysis
→ leaky bilayer → drug
release kinetics → stability
Equipment, conditions
• For filtration, sterilization
• Type, size of filters used
27

28. Points to consider
Role of formulation conditions
Successful development for lipid based RNA delivery and vaccines, Shiksha Mantri, 2020
Characteristics, Stability, PerformanceProcess conditions
DP= drug product
Storage conditions
• Buffer composition, pH,
ionic strength, Storage
vials
Process, conditions
• Cryopreservants
• Inert gas
• Vial type, size, volume
RNA
aqueous
Lipids
Organic
solvent
LNPs
Pre-bulk
LNPs
bulk
Buffer exchange/
dialfiltration/
sterlization
LNPs
Final DP
Lyophilization/
fill & finish
+
Formulation
process
28

29. Points to consider
Role of formulation conditions
Successful development for lipid based RNA delivery and vaccines, Shiksha Mantri, 2020
RNA
aqueous
Lipids
Organic
solvent
LNPs
Pre-bulk
LNPs
bulk
Buffer exchange/
dialfiltration
LNPs
Final DP
Lyophilization/
fill & finish
+
Formulation
process
pH, Temperature, pressure
Lipid concentration
N/P
Storage conditions
DP= drug product
Hold times, process
parameters
Require appropriate analytical methods
to identify critical process parameters
29
Characteristics, Stability, PerformanceProcess conditions

30. Analytical tests required
Successful development for lipid based RNA delivery and vaccines, Shiksha Mantri, 2020
FDA guidance for industry on liposomal drug products, 2018
 Identifying and quantifying drug product
compounds, degradation products
 Lipid identity, impurities, quantity
 Residual solvents
 Parameters of the contained drug
Chemical
Biological
 Sterility
 Endotoxins measurement
30
Physical
 Size, PDI
 pH, osmolality
 Zeta potential
 Phase transition temperature
 Particulate matter

31. Agenda
1. Introduction
Benefits and considerations for RNA therapeutics
2. Critical parameters defining activity of lipid-based formulations
Composition, source, quality, formulation process
3. Essential considerations for successful drug development
Timing, regulatory aspects, number of sources, supplier choice
4. Summary and Q&A

32. 1. Research
composition, formulation process
2. Feasibility studies
process research, gram scale manufacture
3. Process optimization
yield, critical raw materials, stability studies
4. Scale up
analytical methods implementation, cleaning, packaging, safety, GMP runs
5. Process maturation
process validation, risk analysis, intermediates
Timing is key
Synchronize product development with drug development
Successful development for lipid based RNA delivery and vaccines, Shiksha Mantri, 2020
3-15 years for 1 drug approval
Approved drug
FDA approval
Post marketing
surveillance
T= 0
1-6 years
2-7 years
0.5-2 years
Drug discovery
Preclinical
Clinical
trials
Drug discovery
Clinical
trials
Planning product development
32

33. Plan ahead
Tips to accelerate drug approval process
Successful development for lipid based RNA delivery and vaccines, Shiksha Mantri, 2020
✓ Data: Perform all pre-clinical and clinical tests
✓ Products specific documentation:
Descriptions of manuf. process, components, in-
process controls, DMF – not necessary
✓ Consistent quality: changes and their clinical
relevance need description
Before market approval:
✓ Validated processes, analytical methods,
impurities identified and specifications (ICH Q3A
and ICH Q6A guidelines, respectively)
✓ 3 stability studies of each lipid and formulation
Abbreviations used: ICH: International Council for Harmonisation, DMF: Drug master
file, CMC: Chemistry, manufacturing, controls
3-15 years for 1 drug approval
Approved drug
FDA approval
Post marketing
surveillance
T= 0
1-6 years
2-7 years
0.5-2 years
Drug discovery
Preclinical
Clinical
trials
Drug discovery
Clinical
trials
33

34. Characteristics of the right partner
Partners play a big role
1
3
2
High quality products
 High and consistent quality GMP
raw materials
 Formulation services
Deep knowhow
Deep knowledge in:
• Raw material and LNP
manufacturing, analytics
• Regulatory landscape, dossier
preparation
Right facilities
 Appropriate quality systems: Manufacturing under ICH Q7
 Excellent audit track record
 Consistent supply
Successful development for lipid based RNA delivery and vaccines, Shiksha Mantri, 202034

35. Points to consider and risk mitigation strategies
Appropriate number of suppliers
Successful development for lipid based RNA delivery and vaccines, Shiksha Mantri, 2020
✓ Find a supplier who can manufacture out of 2
sites using same process and analytical methods
✓ 3-5 year supply agreements
✓ Establish safety stocks
✓ Maintain a quality agreement
2 or more sources
for each raw material?
Pros: Security of supply
Cons:
• Management of multiple suppliers is time and cost consuming
• Higher regulatory workload
• Additional documents required, e.g. multiple CMC sections
• Detailed description of differences
• Show equivalency of performance
• Increased risk of regulatory hurdles
Risk mitigation
strategies :
1 source/ raw material
35

36. How can we help?

37. + + +=
Special lipid Helper lipids
Custom
manufacturing
Lipid Portfolio
✓ Ionizable lipids
✓ PEG lipids
✓ Targeted lipids
✓ Synthetic origin lipids
✓ Broad portfolio
✓ Customized pack sizes
Fully Support lipid based drug delivery
Product
Development
Preclinical
Collaboration, Innovation, Speed
Development
(Clinical Phases)
On-Time, Scale-Up, Flexibility
Commercial
Consistency – Regulatory - Cost
High
frequented
inspected
sites
CoE & world
leading Supplier
for lipids
All competence on
site with strong
connection to global
expertise
Strong team with
85+ years of
combined
experience
Technical,
analytical,
regulatory
support

38. Agenda
1. Introduction
RNA therapeutics, Comparison of different RNA drug delivery systems
2. Critical parameters defining activity of lipid-based formulations
Composition, source, quality, formulation process
3. Essential considerations for successful drug development
Timing, regulatory aspects, number of sources, supplier choice
4. Summary and Q&A

39. Quality → performance
Summary
1
Plan ahead
Intelligent
formulation design,
Choice of lipids,
source, formulation
process
2
3 Right partners
Reduce cost of drug
development, and
boost success
4
Plug and play
Encapsulation
systems for RNA
therapeutics and
vaccines
Successful development for lipid based RNA delivery and vaccines, Shiksha Mantri, 202039
Ensure quality
Inconsistent quality=
irreproducible results,
regulatory hurdles, high
costs, Wasted resources