Our fourth webinar in the MDC Connects Series 2021 | A Guide to Complex Medicines. This slide deck takes a closer look Lipid Nanoparticles, and how there is so much more to them than being a little fat blob. Yvonne Perrie (University of Strathclyde)

1. Yvonne Perrie et al.
Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde
Lipid nanoparticles:
so much more than a little fat blob

2. https://marlin-prod.literatumonline.com/cms/attachment/8b7c7a39-61b8-4d7c-b099-d20550408343/fx1_lrg.jpg; http://sitn.hms.harvard.edu/wp-content/uploads/2015/05/Fig-12.png
Nucleic acid vaccines
2 Dec 2020
18 Dec 2020 (US)

3. Barriers
RNA are generally active in
cytoplasm
DNA must localize to the
nucleus, where gene expression
cassette is transcribed

4. RNA
• Easily and quickly degraded by RNases
• Highly anionic and hydrophilic, so impairs its cellular uptake.
Ladder
RNA
RNA
+
RNase
https://www.flaticon.com
Delivery

5. Liposomes and LNPs
RNA
Ionisable lipid
(binds the RNA)
Stabilising lipids
LNPs
Liposomes Aqueous core Bilayer membrane

6. What we need from a delivery system
Protection
Potency
Controlled biodistribution
Manufacturability

7. Small Methods, Volume: 2, Issue: 9, First published: 26 April 2018, DOI: (10.1002/smtd.201700375)
Commonly used lipids:
Cationic/ionizable lipids
Apparent pKa
-
6.7
6.4
Bind to the anionic RNA

8. Small Methods, Volume: 2, Issue: 9, First published: 26 April 2018, DOI: (10.1002/smtd.201700375) ; Kim, J., Jozic, A. & Sahay, G. Naturally Derived Membrane Lipids Impact Nanoparticle-Based Messenger RNA Delivery. Cel. Mol. Bioeng. 13, 463–474 (2020).
https://doi.org/10.1007/s12195-020-00619-y
Combine these with Stabiliser lipids:
0%
25%
50%
75%
100%
Molar
ratio
Pegylated lipid
Ionisable/cationic lipid
Cholesterol
DSPC
DLinMC3DMA

9. General LNP production method
Small Methods, Volume: 2, Issue: 9, First published: 26 April 2018, DOI: (10.1002/smtd.201700375)
pH adjusted so that the ionisable lipid is cationic and binds to the RNA
Lipids in solvent
mRNA
The pH is then
raised to 7.4,
resulting in the
outside of the LNPs
being neutral.
LNPs
Toroidal mixer
Staggered herringbone mixer

10. Lipids in
ethanol
Aq phase
Microfluidic production parameters to consider:
Critical Quality Attributes:
Size, PDI
Zeta Potential
Lipid yield
Loading & release profiles
In vivo efficacy
Mixing
ratio
Flow Rate

11. cLNP iLNP
Cationic Lipid Nanoparticles
DSPC:Chol:DOTAP:DMG-PEG2000
DSPC:Chol:DDAB:DMG-PEG2000
Ionisable Lipid Nanoparticles
DSPC:Chol:DLinMC3DMA:DMG-PEG200
Structural/Helper
lipid
DSPC DMG-PEG2000
DOTAP MC3
Chol
DDAB
Cationic/Ionisable
lipid
Molar%
 Structural lipid: 10
 Cholesterol: 48
 Cationic/Ionisable: 40
 PEG lipid: 2
LNP compositions investigated

12. Production: mixing ratio and production rate
Lipids in solvents
Aqueous phase
Flow
rate
ratio
1:1
3:1
5:1
Mixing ratio:
A critical process parameter & formulation dependent.
0.0
0.2
0.4
0.6
0.8
1.0
0
200
400
600
800
1000
DOTAP MC3 DDA DOTAP MC3 DDA DOTAP MC3 DDA
PDI
Z-average
(d.nm)
Size (d.nm) PDI
1:1 3:1 5:1
FRR
All ZP values between 0 and + 4 mV
Manufacturing Considerations for the Development of Lipid Nanoparticles Using Microfluidics. Roces CB, Lou G, Jain N, Abraham S, Thomas A, Halbert GW, Perrie Y.
Pharmaceutics 2020, 12(11), 1095;

13. 0.0
0.2
0.4
0.6
0.8
1.0
0
20
40
60
80
100
DOTAP MC3 DDA DOTAP MC3 DDA DOTAP MC3 DDA
PDI
Z-average
(d.nm)
Production: mixing ratio and production rate
Lipids in solvents
Aqueous phase
Flow
rate
ratio
1:1
3:1
5:1
Mixing ratio:
A critical process parameter & formulation dependent.
Lipids in solvents
Aqueous phase
Flow rate
Production speed:
A critical process parameter & formulation dependent.
0.0
0.2
0.4
0.6
0.8
1.0
0
200
400
600
800
1000
DOTAP MC3 DDA DOTAP MC3 DDA DOTAP MC3 DDA
PDI
Z-average
(d.nm)
Size (d.nm) PDI
1:1 3:1 5:1
FRR
5 mL/min 10 mL/min 20 mL/min
TFR
All ZP values between 0 and + 4 mV

14. Loading is high across lipids and nucleic acids
Range of surrogates can be used for pre-clinical development
0
20
40
60
80
100
DOTAP MC3 DDAB
%
Loading
ionisable/cationic lipid
PolyA ssDNA mRNA
Lipid
choice

15. Groups of ten BALB/c mice were immunized i.m. on days 0 and 28 with either 1.5 or 0.15 μg of self-
amplifying RNA encoding for rabies G protein encapsulating DOTAP polymeric nanoparticles (NPs), DOTAP
Liposomes or DDA Liposomes and compared with the commercial vaccine Rabipur (1/20 of human dose).
Day 0 Day 28
1.5 or 0.15 μg of self-amplifying RNA encoding for rabies
virus glycoprotein G (SAM-RVG) encapsulated
Day 42
Impact on potency
1. DOTAP cLNPs
2. DDA cLNPs
3. MC3 iLNPs

16. Immune profiles: cLNP vs iLNP
 No sig difference between iLNPs and DOTAP and DDA-cLNPs
 All promote anti-RVG IgGs above the correlate of protection
two weeks after a single vaccination.
2 weeks after 1st injection
 titers increased up to 20-fold
 no sig. difference between the cLNP and iLNP responses.
2 weeks after 2nd injection
* (sig higher at lower dose)

17. Biodistribution protocol
IVIS Spectrum In Vivo
Imaging System
Vaccination
Intramuscular
lipid nanoparticles

18. Biodistribution profiles
DOTAP and DDA cLNP –
different
DOTAP cLNP and iLNP –
similar
Does not give insight into preferred biodistribution

19. Manufacturing
parameters
Formulation
parameters
Scale-up

20. Scale-up to GMP
0
20
40
60
80
100
SHM TrM TrM 60 mL/min TrM 200
mL/min (GMP)
Encapsulation
Efficiency
(%)
200
60
12
12
12 200
60
12
12
mL/min mL/min
0
0.2
0.4
0.6
0.8
1
0
20
40
60
80
100
GenVoy-ILM™
(empty)
SHM TrM TrM 60 mL/min TrM 200
mL/min (GMP)
PDI
Z-average
(d.nm)
Size PDI

21. Summary

PHA-ST-TRAIN-VAC (EU Hoizon2020)
Despo Chatzikleanthous
Giulia Anderluzzi
Gustavo Lou Ramirez
Rob Cunliffe
EPSRC CDT
Cameron Webb
Gillian Berrie
Neil Forbes
KTP (Lamellar Biomedical)
Rachel Donaghey
Micorsun (Innovate UK)
Carla Roces
Independent Research Fund Denmark
Signe Schmidt