Translating the production of nanomedicines from bench to GMP - Yvonne Perrie et al. Strathclyde Institute of Pharmacy and Biomedical Sciences ...
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Translating the production of
nanomedicines
from bench to GMP
Yvonne Perrie et al.
Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde
Nanomedicines.
Challenges – includes efficient and robust manufacture
Research Market
Lots of evidence at Scalability & manufacture
lab scale
Applies to old and new:
£
“Valley of death”
Research objective - develop a manufacturing strategy
Research
Lots of evidence at lab scale Responsive & scalable
manufacture
Microfluidic production - options
Example of microfluidic mixers:
a) Toroidal mixer b) Staggered herringbone mixer c) Basic T-mixer d) Hydrodynamic flow focussing (2D and 3D)
Aqueous buffer phase Operating parameters
Flow rate ratio/mixing ratio: the ratio that the aqueous and non-aqueous streams are
Solvent phase containing phospholipids
mixed at (normally stated as a v:v ratio).
Liposome product Total flow ratio/operating ratio: total running speed (normally in mL/min).
Shah et al., 2020. Liposomes: Advancements and innovation in the manufacturing process, submitted
Liposomes and LNPs
Negative Neutral Positive
- +
- - + +
- - + +
- +
Common Drugs Drugs Drugs
vehicle for: Subunit antigens Subunit antigens Subunit antigens
DNA, RNA (LNPs)
Action(s): Protection,
Delivery & Targeting
Adjuvant Lipid nanoparticles1
1Image from Gustavo Lou, 2020 thesis: Design of novel delivery systems to probe alternative routes of administration for a self-amplifying RNA-based rabies vaccine (https://pureportal.strath.ac.uk/en/studentTheses/design-of-novel-
delivery-systems-to-probe-alternative-routes-of-a)
Scaling to GMP
Scaling up production - capacity
-
- -
- -
- +
+ +
Flow rate
capacity +
+
+
1 – 20 mL/min
Staggered Herringbone mixer (SHM)
High flow rate
capacity
> 20 L/h
Toroidal mixer (TrM)
Using microfluidics for scalable manufacturing of nanomedicines from bench to GMP: A case study using protein-loaded liposomes. Webb C, Forbes N, Roces CB, Anderluzzi G, Lou G, Abraham S, Ingalls L,
Marshall K, Leaver TJ, Watts JA, Aylott JW, Perrie Y. Int J Pharm. 2020 Apr 3;582:119266. doi: 10.1016/j.ijpharm.2020.119266. [Epub ahead of print]
ü Mapping across the mixer design:
SHM TrM
C) D)
A) SHM B) SHM 100 100
Percentage release (%)
80 80
Lipid percent (%)
60 60
40 40
SHM
20 20 TrM
100 nm 100nm
100 nm Free OVA
0 0
Before microfluidics After microfluidics 0 20 40 60 80 100 120
Time (hours)
Performed on neutral liposomes incorporating OVA
Using microfluidics for scalable manufacturing of nanomedicines from bench to GMP: A case study using protein-loaded liposomes. Webb C, Forbes N, Roces CB, Anderluzzi G, Lou G, Abraham S, Ingalls L,
Marshall K, Leaver TJ, Watts JA, Aylott JW, Perrie Y. Int J Pharm. 2020 Apr 3;582:119266. doi: 10.1016/j.ijpharm.2020.119266. [Epub ahead of print]
Substances Process step In-process and quality controls
Lipids in m/ethanol Manufacture using IPC
microfluidics 1. Liposomes attributes (DLS, Malvern AT):
API in buffer a) particle size,
b) PDI,
c) zeta potential.
2. Lipid recovery (HPLC-ELSD).
Aq buffer Purification using IPC
cross-flow filtration 3. Solvent removed (GC).
4. Free API removed (HPLC).
5. Liposome recovery (HPLC-ELSD or DilC).
6. Liposome size, pdi and zeta potential
(DLS, Malvern AT).
7. API loading (HPLC).
QC
Testing to final product
specification
Scale up to GMP (12 to 200 mL/min)
A) 100 1.0 B) 60 C) 50 -7 ± 1 mV -9 ± 2 mV -8 ± 1 mV
Size
Z-average diameter (nm)
80 PDI 0.8 40
Protein Loading (%)
200 mL/min
Intensity (A.U)
40
60 0.6 30
PDI
40 0.4 60 mL/min 20
20
Ignite
Blaze
GMP
20 0.2 10
12 mL/min
0 0.0 0 0
0 100 200 5 50 500 5000 12 60 200
Total Flow Rate (mL/min) Z-average diameter (nm) Total flow rate (mL/min)
Performed on neutral (DSPC:Chol) liposomes incorporating OVA @ ambient temp
Using microfluidics for scalable manufacturing of nanomedicines from bench to GMP: A case study using protein-loaded liposomes. Webb C, Forbes N, Roces CB, Anderluzzi G, Lou G, Abraham S, Ingalls L,
Marshall K, Leaver TJ, Watts JA, Aylott JW, Perrie Y. Int J Pharm. 2020 Apr 3;582:119266. doi: 10.1016/j.ijpharm.2020.119266. [Epub ahead of print]Process parameters to consider
16 Small
Large
Critical process parameters: Buffer
12
Intensity (%)
8
4
0
1 10 100 1000 10000
Hydrodynamic size (d.nm)
A) Production of SUV
Lipid mix
Solvent removal
10 mM TRIS
Small (40 nm) cationic liposomes Small (40 nm) cationic 200 nm
in 10 mM TRIS liposomes (10 mM TRIS)
B) Production of LUV
Lipid mix
Solvent removal
High conc TRIS Buffer exchange
Large (>500 nm) cationic liposomes Large (>500 nm) cationic
in high buffer concentration liposomes (10 mM TRIS)
A novel microfluidic-based approach to formulate size-tuneable large unilamellar cationic liposomes: Formulation, cellular uptake and biodistribution investigations. Lou G, Anderluzzi G, Woods S, Roberts CW, Perrie
Y. Eur J Pharm Biopharm. 2019 Oct;143:51-60. doi: 10.1016/j.ejpb.2019.08.013. Epub 2019 Aug 22.CPP: Solvent and mixing rate
80 1
size PDI 0.8
60
Size (nm)
0.6
40
PDI
0.4
20
0.2
0 0
1:01 3:01 5:01
Flow rate ratio
The Impact of Solvent Selection: Strategies to Guide the Manufacturing of Liposomes Using Microfluidics. Webb C, Khadke S, Schmidt ST, Roces CB, Forbes N, Berrie G, Perrie Y. Pharmaceutics. 2019 Dec
4;11(12). pii: E653. doi: 10.3390/pharmaceutics11120653.Responsive scale-independent production confirmed
Case studies Research Responsive & scalable
manufacture
-
- -
Anionic formulations for
lymphatic targeting
- -
-
+
ü ü
+ +
Cationic liposomes for
vaccine delivery
+ +
+Delivery and Targeting strategies Targeting the lymphatics
Charge effects PK of liposomes
Formulations Particle size Zeta Potential
(nm) (mV)
DSPC:Cholesterol 101 ± 1 -2 ± 0.6
DSPC:Cholesterol:PS 90 ± 7 -66 ± 4
DSPC:Cholesterol:DOTAP 110 ± 4 62 ± 5
100 40
DSPC:Cholesterol
% Dose @ lymph node
DSPC:Cholesterol
% Dose a@ injection site
80 DSPC:Cholesterol:PS DSPC:Cholesterol:PS
30
DSPC:Cholesterol:DOTAP DSPC:Cholesterol:DOTAP
60
20
40
10
20
0 0
0 50 100 150 200 0 50 100 150 200
Time (h) Time (h)
Formulation and manufacturing of lymphatic targeting liposomes using microfluidics. Khadke S, Roces CB, Cameron A, Devitt A, Perrie Y. J Control Release. 2019 Aug 10;307:211-220. doi:
10.1016/j.jconrel.2019.06.002. Epub 2019 Jun 3Improving lymph node targeting • Consider using active-targeting mechanism • Biotin Avidin complex
Biotin Avidin complex
1. Pre-dose with avidin 2. Then inject biotin liposomes 3. Avidin-biotin liposome
(30 min) complex forms
Lymph nodeAnionic-biotin formulations
Formulations Lipid ratio (µmoles) Particle PDI Zeta Antigen
size (nm) Potential loading
(mV) (%)
DSPC:Cholesterol:PS 6:4:2.5 113 ± 2 0.15 ± 0.02 -41 ± 5 19 ± 4
DSPC:Cholesterol:PS:DSPE-
6:4:2.5 + 20 mole% 88 ± 7 0.24 ± 0.02 -12 ± 2 10 ± 3
PEG 2k biotin (20 mole %)
Formulation and manufacturing of lymphatic targeting liposomes using microfluidics. Khadke S, Roces CB, Cameron A, Devitt A, Perrie Y. J Control Release. 2019 Aug 10;307:211-220. doi:
10.1016/j.jconrel.2019.06.002. Epub 2019 Jun 3Re-directing to the lymph nodes
Site of injection: Popliteal lymph node: Inguinal lymph node:
100 40 40
% dose @ injection site
Liposomes
DSPC:Chol:PS
80 30 30 No notable
% dose at POP
% dose at ILN
Protein (H-56)
Antigen
60 difference
20 20
40 in immune
20 10 10 responses.
0 0 0
0 100 200 0 100 200 0 100 200
Applications
Time (h) Time (h) Time (h) in drug
delivery.
DSPC:Chol:PS:DSPE-PEG 2k
Biotin (20 mole%)/avidin
100 40 40
% dose @ injection site
80 30 30
% dose at POP
% dose at ILN
60
20 20
40
20 10 10
0 0 0
0 100 200 0 100 200 0 100 200
Time (h) Time (h) Time (h)Cationic formulations for vaccines Improved delivery and adjuvant action
Application of cationic liposomes in vaccines:
enhancing efficacy of protein-CpG conjugates
Group B Streptococcus (GBS) GBS67 protein
antigen with the CpGODN TLR9 agonist
Design of a novel vaccine nanotechnology-based delivery system comprising CpGODN-protein conjugate anchored to liposomes. Chatzikleanthous D, Schmidt ST, Buffi G, Paciello I, Cunliffe R, Carboni F, Romano MR, O'Hagan DT,
D'Oro U, Woods S, Roberts CW, Perrie Y, Adamo R. J Control Release. 2020 Apr 2. pii: S0168-3659(20)30211-X. doi: 10.1016/j.jconrel.2020.04.001. https://t.co/aE5YyJyCen?amp=1Immunological evaluation of the designed system
Day 0 Day 21 Day 42
Immunisation Immunisation Isolation of spleens
Route: IM
Antigen dose: 1 μg
CpG dose: 0.15 μg
Liposome dose: 50 μg
Group 1 Group 2 Group 3 Group 4 Group 5 Group 6
Protein Conjugate Antigen/Liposome Conjugate/Liposome Mixture/Liposome
Mixture
All experiments were undertaken in accordance with the regulations of the Directive 2010/63/EU.
Group B Streptococcus (GBS) GBS67 protein antigen with the CpGODN TLR9 agonistImmunogenicity boasted by cationic liposomes:
Mean Log reciprocal endpoint titres ± SD
The combination of the conjugate and
the liposomes provided significantly
higher responses for Total IgG, IgG1
and IgG2a.
Antibodies confirmed to be
functional.
Similar story for cytokines tested:
Group 6
Pre-immunisation Group 1
Protein
Group 2
Conjugate
Group 3
Mixture
Group 4
Antigen/
Group 5
Conjugate/ Mixture/ IFNg, IL-13, IL-17A, IL-21, IL-22, IL-9,
Liposome
Liposome Liposome
IL-10
Total IgG responses after boost dose (day 42). Six groups of mice were injected twice intramuscularly with the corresponding formulations. The study was split over two experiments with 2 mice from each group in study 1 and 3
mice in study 2. The results are then combined to give an n = 5. Results are plotted for individual mouse and also an average, so show variability across the studies and mice. Blood samples were taken from the tail at day 21. Mice
were terminated at day 42 and ELISA was performed for determination of total GBS67-specific antibody titre levels. Mixture and conjugate are represented by (+) and (-), respectively. Results are the average of two independent
experiments (mean ± SD). ***pCationic liposomes promote depot Biodistribution profile of protein and liposomes. (A) Whole-body fluorescence intensity images for all groups for selected days. (B) Protein and (C) liposomes dose retention at the site of injection following intramuscular. injection of either free GBS67, GBS67 conjugated to CpGODN (GBS67-CpGODN) or GBS67-CpGODN adsorbed on the surface of DSPC: Cholesterol: DDA cationic liposomes (GBS67-CpGODN+Liposomes). Mice received 10 μg protein-based dose of vaccine, corresponding to the administration of 1.5 μg of TLR9 agonist. A 200 μg dose of cationic liposomes was used in one of the groups. A naive mouse was used as negative control. The proportion of Alexa Fluor 790 tracking dye at the injection site as a percentage of the initial dose was calculated. Mixture and conjugation are represented by (+) and (-), respectively. Dash line on Figure (C) represents the background level. Results represent the mean ± SD of three mice per group. ***p
Summary
Nanomedicines PHA-ST-TRAIN-VAC
Microfluidics Despo Chatzikleanthous
ü Drug/antigen protection ü Responsive Giulia Anderluzzi
ü Drug/antigen delivery ü Reproducible Gustavo Lou Ramirez
ü Improved potency ü Scale-independent Rob Cunliffe
EPSRC CDT
Cameron Webb
ü Gillian Berrie
Maryam Hussain
Neil Forbes
KTP (Lonza)
Swapnil Khadke
KTP (Lamellar Biomedical)
Rachel Donaghey
Micorsun (Innovate UK)
Carla Roces
Independent Research Fund Denmark
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