prof ian marison, director, national institute for bio-processing research & training, nibrt

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?