W H I C H PA R A M E T E R S U S E T O O B TA I N G O O D P H Y S I C O C H E M I C A L C H A R A C T E R I S T I C S ?
Noémie Penoy
PhD Student
npenoy@uliege.be
CENTER FOR INTERDISCIPLINARY RESEARCH ON MEDICINES 2
LIPOSO
ME
Encapsulation of hydrophilic/hydrophobic molecules Targeting specific tissues Increased efficiency Decreased toxicity Protection of the active molecule
Classical laboratory methods to prepare
liposomes
Thin film hydration
Detergent removal
Solvent injection
Reverse phase evaporation
Emulsion method
TFH : method used in our laboratoryCENTER FOR INTERDISCIPLINARY RESEARCH ON MEDICINES 4
Classical laboratory methods to prepare
liposomes
Lots ofdrawbacks
Use of organic solvents Small quantities
produced
Several steps fo form liposomes
No GMP conditions
W. Bigazzi, N. Penoy et al. , Supercritical fluid methods: An
alternative to conventional methods to prepare liposomes, Chem. Eng. J., October 2019
Innovative methods to prepare liposomes
Microfluidic method
Homogenization method
CENTER FOR INTERDISCIPLINARY RESEARCH ON MEDICINES 6
Advantages :
Solvating properties
Miscibility with organic solvents High density
Viscosity and diffusivity similar to gases
Promotes mass transfer
Particles design/Drug formulation
Supercritical carbon dioxide
W. Bigazzi, N. Penoy et al. , Supercritical fluid methods: An
alternative to conventional methods to prepare liposomes, Chem. Eng. J., October 2019
Particles design/Drug formulation
Supercritical carbon dioxide
used as :
Solvent Cosolvent Antisolvent
Dispersing agent
CENTER FOR INTERDISCIPLINARY RESEARCH ON MEDICINES 8
Supercritical CO
2as a dispersing
agent
Lipids in aqueous buffer Reactor sc CO2W. Bigazzi, N. Penoy et al. , Supercritical fluid methods: An
alternative to conventional methods to prepare liposomes, Chem. Eng. J., October 2019
Reactor Lipids in aqueous buffer in supercritical conditions Legend Phospholipid Supercritical CO2 Aqueous buffer
Supercritical CO
2as a dispersing
agent
Reactor Lipids in aqueous buffer in supercritical conditions sc CO2 Lipids organize in bilayersCENTER FOR INTERDISCIPLINARY RESEARCH ON MEDICINES 10 Reactor sc CO2 Lipids in aqueous buffer in supercritical conditions Reactor Liposomes suspension Legend Phospholipid Supercritical CO2 Aqueous buffer
Supercritical CO
2as a dispersing
agent
W. Bigazzi, N. Penoy et al. , Supercritical fluid methods: An
alternative to conventional methods to prepare liposomes, Chem. Eng. J., October 2019
Supercritical CO
2as a
dispersing agent
Without any use of organic solvent
In one step
Large scale production
CENTER FOR INTERDISCIPLINARY RESEARCH ON MEDICINES 12
In practice lots of parameters are involved in
the process
Process parameters
Pressure (bars) Temperature (°C) Agitation rate (rpm) Contact time (h)Formulation parameters
Phospholipids concentration (mM) Suspension volume (mL)(CQAs)
For drug delivery applications :
Size < 200 nmCENTER FOR INTERDISCIPLINARY RESEARCH ON MEDICINES 14
Find the optimal conditions for liposome production
using supercritical CO
2by a QbD approach
Find conditions allowing the production of liposomes of desired features (± 200 nm)
1. Formulation Soy phosphatidylcholin e (SPC) (70%) (m/m) Cholesterol (CHOL) (30%) (m/m) 2. Sample preparation 65°C 1200 rpm 15 min 3. Supercritical CO2 process Lipids in aqueous buffer in supercritical conditons Liposomes suspension Legend Phospholip id Supercritical CO2 Aqueous buffer
CENTER FOR INTERDISCIPLINARY RESEARCH ON MEDICINES 16
Liposome size (nm) and dispersity analysis by Dynamic Light Scattering (n=3)
Desired answers: Size ± 200 nm Exp Lipids concentration (mM ) Volum e (mL) Contact time (h) Agitation rate (rpm) Temperat ure (°C) Pressu re (bars) Bloc k 1 51,0328 10 0,5 400 57,5 250 1 2 27,532 20 1 550 57,5 185 1 3 50,5127 30 2 700 35 250 1 4 5,05833 10 2 400 80 120 1 5 5,007053 30 2 400 35 120 1 6 50,0675 30 0,5 400 80 250 1 7 50,03196 10 0,5 400 80 120 1 8 27,5644 20 1 550 57,5 185 1 9 5,08813 30 0,5 700 57,5 120 1 10 5,02385 30 2 400 80 250 1 11 5,0671 30 0,5 400 35 250 1 12 5,02021 10 0,5 700 35 250 1 13 50,8555 10 0,5 700 35 120 1 14 50,3337 10 2 700 57,5 250 1 15 5,3016 10 2 700 80 250 1 16 50,08459 10 2 400 35 185 1 17 4,9426 10 0,5 700 80 185 1 18 49,9899 30 0,5 700 80 185 1 19 50 30 2 700 80 120 1 20 27,52 20 1,25 550 57,5 185 1 Exp Lipids concentration
(mM ) e (mL)Volum time (h)Contact
Agitation rate
(rpm) Temperature (°C) e (bars)Pressur Block
1 30,0008648 15 0,5 600 80 200 2 2 29,9407 15 0,5 600 50 120 2 3 5,05938 15 0,5 400 59 120 2 4 5,03745 30 0,5 600 80 120 2 5 4,9897 30 0,5 400 80 160 2 6 29,9572 30 0,5 400 60,35 120 2 7 5,01582 15 0,5 400 50 200 2 8 5,08151 30 0,5 600 62,6 176,4 2 9 4,9383 30 0,5 400 57,95 181,2 2 10 30,08587 15 0,5 600 80 200 2 11 30,0433 30 0,5 400 56,6 175,6 2 12 17,5542 22,5 0,5 500 65 160 2 13 17,6341 22,5 0,5 500 65 160 2 14 17,5967 22,5 0,5 500 65 160 2
Lipid Concentration (mM) Agitation Rate (rpm) Temperatur e (°C) Pressure (bars) Contact Time (h) Suspension Volume (mL) S iz e (n m ) P d I Z e ta Po te n ti a l (m V ) 27.5 mM 20 mL 1.25 h 550 rpm 57.5 °C 185 bars D e si ra b ili ty Non influential parameters : Contact time : 30 minutes Agitation rate : 500 rpm Influential parameters : Lipids concentration (mM) Suspension volume (mL) Temperature (°C) Pressure (bars)
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: High defect rate : Low defect rate
Condition 1
Condition 2
Concentration : 45 mM Volume : 14 mL Temperature : 80 °C Pressure : 240 bars Expecting values : 51-213 nm Concentration : 5 mM Volume : 10 mL Temperature : 80 °C Pressure : 156 bars Expecting values : 67-221 nm SCALE-UPCENTER FOR INTERDISCIPLINARY RESEARCH ON MEDICINES 20 Formulation SPC/CHOL 70/30 % (m/m) : n = 3 Condition 1 Condition 2 45 mM 5 mM 14 mL 10 mL 80°C 80°C 240 bars 156 bars
Are these conditions
transferrable to other
liposomes formulations ?
CENTER FOR INTERDISCIPLINARY RESEARCH ON MEDICINES 22 Formulation SPC/CHOL/DSPE PEG 2000 65/30/5 % (m/m) Condition 1 Condition 2 45 mM 5 mM 14 mL 10 mL 80°C 80°C 240 bars 156 bars n = 3 Cond 1 Cond 2 0 50 100 150 200 250 S iz e (n m )
Formulation EPC/DC-CHOL/CHOL/DSPE PEG 2000 50,05/29,4/0,6/19,95 % (m/m) Condition 1 Condition 2 45 mM 5 mM 14 mL 10 mL 80°C 80°C 240 bars 156 bars n = 3 0 100 200 300 S iz e (n m )
CENTER FOR INTERDISCIPLINARY RESEARCH ON MEDICINES 24 Condition 1 Condition 2 45 mM 5 mM 14 mL 10 mL 80°C 80°C 240 bars 156 bars n = 3 Formulation DOPE/CHEMS/CHOL/DSPE PEG 750 43/21/30/6 % (m/m) 0 100 200 300 400 S iz e (n m )
Formulation DOTAP/CHOL/DOPE 44,45/33,33/22,22 % (m/m) Condition 1 Condition 2 45 mM 5 mM 14 mL 10 mL 80°C 80°C 240 bars 156 bars n = 3 Cond 1 Cond 2 0 200 400 600 S iz e (n m )
CENTER FOR INTERDISCIPLINARY RESEARCH ON MEDICINES 26 Condition 1 Condition 2 45 mM 5 mM 14 mL 10 mL 80°C 80°C 240 bars 156 bars
The influential parameters of the process are : the lipids concentration
the suspension volume the temperature
the pressure
2 working conditions
Transferrable to simple formulation Optimization
Condition 1 promising for the scaling-up ± 200 nm
Suppress the sample preparation (dispersion step) One step process
Understand the effect of supercritical conditions on liposome formation
Integrity of the phospholipids by NMR
Modification of the glass transition temperature using DSC
CIRM ULiège Coralie Bellefroid Olivier Jennotte Nathan Koch Noémie Penoy Isaïe Niamba Luc Delma Ange Ilangala Chloé Parulski Manon Berger Laurence Collard Françoise Léonard Vanessa Strauven
