prof ian marison, director, national institute for bio-processing research & training, nibrt
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Encapsulation as a method for non-parenteral drug and cell delivery
Prof. Ian W. MarisonLaboratory of Integrated Bioprocessing (LiB)
Dublin City UniversityNational Institute of Bioprocessing Training & Research
(NIBRT)
Presentation outline
• Introduction: an example of an innovative NIBRT research programme
• High cell density cultures by cell encapsulation
• Microcapsule characterisation
• Antibiotic encapsulation (geldanamycin)
• NSAID encapsulation
• And what about drug recovery from drinking water?
Biologicals – new challenges
Complex compounds need an holistic, integrated approach
Developing the Nation's Biosimilars ProgramSteven Kozlowski et al. N Engl J Med 2011; 365:385-388 August 4, 2011
Training and research for Industry: transforming performance through constructive partnership
An Innovative Partnership
• National Institute for Bioprocessing Research and Training
• Created for industry – in partnership with industry
• Initially four leading academic institutions- expanding to become a truly National facility e.g. incorporation of all 7 universities and Institutes of Technology
• Funded (€57 million) by the Irish Government (IDA Ireland)
• Operated as a non-profit making company
NIBRT research: creating a competitive advantage
Expression systems
Expression systems for the production of biopharmaceuticals in US & EU
39%
15%
1%
16%29%
E. coli
Yeast
Other microbial cells
CHO
Other animal cells
Figures from ”Expression systems for product and process improvements”, Ronald A. Rader, BioProcess International, June 2008
Challenges of animal cell culture
• Solutions:– Cell Encapsulation
– Process Analytical Technology
• Need for high cell density cultures
• Need for high level of monitoring & control
Cell Microencapsulation
Semi-permeable capsule membrane
Viable cells
Shear stress Wastes
Nutrients
Critical for the survival of the cells
bioreactor
ProliferateRecombinant Protein
Capsule(Micro-bioreactor)
Vibrating-Jet Technique
Size range 150 μm - 2 mm and deviation of ± 1.5%
Liquid-core
Porous membrane
Size range 200 μm - 2 mm and deviation of ± 2.5%Whelehan and Marison (2010). Journal of Microencapsulation 28: 669-688
Aqueous two-phase system– PEG and Dextran
• Present work• Obtaining required characteristics• Cell Encapsulation
• Cells suspended within core• Testing new polymers
Development of Novel Microcapsules
Hydrogelmembrane
• Potential impact• High commercialization
possibilities
Dextran in Polymer
Preliminary experiment to optimize cell number in alginate microcapsules
Encapsulation of CHO cells
Empty PLL-alginate microcapsules
CHO 320 Cells 104 / ml alginate 0 0.5 1 2 2.7
AVD Micro capsule (μm) (n=25 microcapsules) 321 +/- 7 335 +/-6 320+/-7 402 +/- 64 381 +/- 48
0.5x104 cells/ml alginate 1x104 cells/ml alginate
2x104 cells/ml alginate 2.7x104 cells/ml alginate
Regular shaped and intact microcapsules
Irregular shaped microcapsules
Difficulties in jet break up
*
*Breguet, V. et al. (2007). Cytotechnology 53: 81-93
Removal of desired compounds from their associated environments
Extraction aides for biotechnological and chemical processes
• Liquid-core• Hydrophobic material• Oleic acid, vegetable oils etc
• Alginate hydrogel membrane• Chitosan, cellulose sulphate etc• Porous structure
• Novel approach termed ‘Capsular Perstraction’
Capsular Perstraction Derived from permeation and extraction
Microcapsules
Further treatment
LiB
Geldanamycin
• Polyketide antibiotic
• Ansamycin family
• Streptomyces hygroscopicus var geldanus
• Received significant attention in 1980’s
• Novel antitumor antibiotic
Pelleted growth(magnification 40X)
LiB
Commercialgeldanamycin
Molecular structureof geldanamycin
ISPR of Geldanamycin
Whelehan & Marison (2011). Biotechnology Progress 27: 1068-1077 & Whelehan et al (2012) Journal of Bioscience and Bioengineering (in press)
• 30% increase in geldanamycin• 110 – 143 mg/l
• Removal from the hostile culture environment• Selectivity
• Downstream processing• Reduced no. of steps• Highly purified
No capsules Biomass growth
GA conc.
0
0.2
0.4
0.6
0.8
1
1.2
0 20 40 60 80
time (min)
C(t
)/C
(0)
485 μm598 μm751 μm
Rapid extraction of the antibioticfrom degradation environment
• Polyketide antibiotic• Ansamycin family
• Streptomyces hygroscopicus var geldanus• Novel antitumour activity
• Produced at relatively low conc.• Sensitive to process conditions
• Liquid-core microcapsules• Increase productivity
Purification of Capsular Geldanamycin
Geldanamycin loaded Capsules (selective removal) Very pure solution
for purificationEmpty capsules for future use
+
Mix with acetonitrile saturated with oleic acid
Agitate at high speed
Geldanamycin, acetonitrile and small quantities of oleic acid
Removal of oleic acid
Low temperaturedistillation
Acetonitrile removal
Crystal production
Geldanamycin crystals (purity > 97%)
Solution has a higher affinity
Application in other fields
• Mechanism to degraded the extracted pollutant
LiB• Methodology for the treatment of drinking/waste-water
• Pharmaceuticals, pesticides and herbicides
Pollutant loadedcapsule
Pseudomonas
0
20
40
60
80
100
120
0 20 40 60 80 100 120
time (min)
% r
emo
ved
Ethylparathion
Methylparathion
Atrazine
2,4 D
0
20
40
60
80
100
120
0 20 40 60 80 100 120time (min)
% r
em
ov
ed
Sulfamethoxazole
Metoprolol
Furosemide
Clofibric Acid
Carbamazepine
Warfarin
Diclofenac
Whelehan et al (2010). Water Research 44:2314-24 Wyss et al (2004). Biotech Bioeng 87:734-42
Determining characteristics of microcapsules• Porosity
– HPLC with dextran standards
HPLC Chromatogram • Mechanical resistance (strength)– Texture analyzer
• Burst Force
Before compression After compression
Atomic Force Microscope (AFM)
Confocal Scanning Laser Microscope (CSLM)
Light Microscope
Techniques for capsule characterisation
Data analysis & Management
Scanning Electron Microscope(Cryo FE-SEM)
633nm laser
DUAL STAINING OF LOW GRADE ALGINATE POWDER
488nm laser
Combined image
Polyphosphates- yellowAlgin -blueBar 5mm
MONITORING POLYMER STRUCTURAL CHANGES - STAGES
Micro-tensile (Deben UK) Shearing
Heating/cooling(CO2/ N2) Heating/cooling (Peltier)
Warm stage
0
100
200
300
400
500
600
700
800
0 20 40 60 80 100 120
Time (sec)
G' (
Pa)
Rheology of slow-induced alginate gel
Alginate powderStored at 55% RH
Poly-phosphate crystals
500 nm
Alginatecharacterisation
0
20
40
60
0 90 180 270 360
position (deg)
coat
th
ickn
ess
(μm
) Membrane thickness 39± 2μm
Confocal MicroscopyAlginate-Poly-L-Lysine-Alginate microcapsules
ATOMIC FORCE MICROSCOPY
• “Feels” surfaces
• Z-resolution ~1 angstrom
• Surface topography
• Force measurements
• Viscoelastic properties
ATOMIC FORCE MICROSCOPYChitosan7 nm spacing
Alginate molecules on capsule surface
Height Phase
Height Phase
AFM - Aqueous-core microcapsulesPRE-LIQUEFACTION
POST-LIQUEFACTION
Post-Liquefaction – Surface topography
208 um
Characterisation of Encapsulated CHO Cells
… 4 × 105 CFUencapsulation
Asylum Asylum MFP-3DMFP-3D
Day 0
Day 3
Are there cells protruding on the capsules surface?
No cell visible on micro-capsule surface
Analysis Mode
Day 4
AFM illustrates cell- polymer interaction within the capsule core
CHO 320 polymer
Analysis Mode
Asylum Asylum MFP-3DMFP-3D
Are the cells embedded in the capsule core?
Encapsulated LIVE CHO DP12
SINGLE METABOLISING CHO 320
CHO 320 EncapsulationLIVE/DEAD analysis
x10
x20
2 × 104 CFU 4 × 105 CFU 5 × 106 CFU
Encapsulation of CHO 320 Confocal Microscopy
… 4 h after CHO cell encapsulation
… 3 × 106 CFUencapsulation
Metabolising CHO cells
after 4 days
Storage of encapsulated CHO 320
SCANNING ELECTRON MICROSCOPY
SCANNING ELECTRON MICROSCOPY
“Oral Delivery of NSAIDs within the gastrointestinal (GI) tract to improve systemic bioavailability, to reduce side effects and to target release to regions of the GI tract to maximise systemic absorption or enable localised delivery to diseased GI tissue”
Thanks to: Bernard McDonald:Joint funded by Sigmoid Pharma and IRCSET
Introduction
40
Opportunities• Once Daily Dosing • Lower Dose • Less Side Effects• Targeted Colonic delivery• ↑ Bioavailability
Enhanced Drug Solubility• Drugs available in solubilised form• ↑ Bioavailability
Enhanced Drug Permeability• Drug passes into bloodstream• Convert injection into oral• ↑ Bioavailability
Enhanced Drug Stability• Controlled/Targeted release
(Polymer coatings)
Encapsulation Technology – Opportunity to address all issues
• Small Molecules• Large Molecules• Peptides
Introduction
41
Capsules
Encapsulation Approaches
Oil Droplets Encapsulated in Gelatin Matrix
API dissolved in oil core, surrounded by gelatin (or other material) shell
Oil Core
Gelatin Shell
API dissolved in oil/surfactant/co-solvent mixture entrapped as droplets in a gelatin (or other material) matrix
Beads
Introduction
42
Model Drug Selected: Celecoxib
• NSAID
• Poorly soluble
• COX-2 inhibitor (Cyclooxygenase-2 plays a role in inflammation)
• Typical indications: osteoarthritis, rheumatoid arthritis, acute pain
• Other indications: role in colorectal cancer prevention (reduces number of colon and rectal polyps) and possible role in colon cancer therapy. Large scale studies have been hindered by side effects
Results – Liquid Formulations
43
25 liquid formulations prepared containing celecoxib dissolved in combinations of oils/surfactants/co-solvents and assessed via in-vitro dissolution testing
Celecoxib Liquid Formulations produced using Optimal Liquid Vehicles
In-vitro Dissolution Testing
• Media maintained at 37 °C
• Paddle speed – 75 RPM
• Automatic sampling over 12 hours
• Use media to replicate intestinal conditions- Simulated gastric fluid (pH 1.2)- Simulated intestinal fluid (pH 6.8)
• All conditions chosen to replicate in-vivo conditions
• Dissolution testing referred to as release testing in the case of pre-dissolved dosage forms
Results – Liquid Formulations
44
Dissolution Performance of Celecoxib API and Marketed Celecoxib Product Celebrex® compared to that of selected Liquid Formulations
• Formulation CEL-021/L superior to Celebrex™ and API• Formulation CEL-021/L superior to CEL-026/L. Drug fully dissolved in both formulations therefore composition very important
45
Results – Optimised Microcapsule Formulations
Release of drug from optimised formulations in excess of 80%
% of release dropped off after 12hrs. Need to apply controlled release polymers to avoid drop-off
Performance of optimised formulations superior to Celebrex™
Formulation CEL-136/B superior to CEL-135/B via incorporation of greater surfactant levels
46
Results – Physical Characterisation of Microcapsules
Correlation between Internal Structure and In-Vitro Performance
Large oil droplets = poor dissolution = likely poor bioavailability
Small oil droplets = good dissolution = likely good bioavailability
• Encapsulation of bioactives• Functional foods
Global f unctional f oods marketGlobal f unctional f oods market
• Global market size ~$75 billion (GBA, 2007) USA >$20 billion; EU, Japan
8% growth globally; 14% USA
key segments: probiotic dairy ~$12 billion, 7% growth thru 2010; omega-3 ~$3 billion, 10% growth)
• Global market size forecast >$100 billion by ~2010
• Encapsulation of Folates• Improved
• Stability of sensitive molecules (digestive system)
• Storage conditions i.e. handling
• Applicability etc
• Enterprise Ireland commercialization grant
Conclusions
• There are a number of innovative programmes in Ireladn in the area of drug delivery of small and large molecules
• NIBRT would be an ideal vehicle for helping to coordinate some of these activities
• Novel technologies exist for drug and cell delivery
• Novel technologies exist for drug and organics recovery
• Potential business opportunities exist to exploit these
• And what about drug recovery from drinking water?
Feel free to contact me:
Prof Ian Marison Executive Director ian.marison@nibrt.ie
Visits can be arranged at any time.
Thank you for your attention!
Any questions?
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