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
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Prof Ian Marison, Director, National Institute for Bio-processing Research & Training, NIBRT
- 1. Encapsulation as a method for non- parenteral drug and cell delivery Prof. Ian W. Marison Laboratory of Integrated Bioprocessing (LiB) Dublin City University National Institute of Bioprocessing Training & Research (NIBRT)
- 2. 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?
- 3. Biologicals – new challenges Developing the Nation's Biosimilars Program Steven Kozlowski et al. N Engl J Med 2011; 365:385-388 August 4, 2011 Complex compounds need an holistic, integrated approach
- 4. Training and research for Industry: transforming performance through constructive partnership
- 5. 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
- 6. NIBRT research: creating a competitive advantage
- 7. Expression systems Expression systems for the production of biopharmaceuticals in US & EU E. coli Yeast Other microbial cells 39% CHO Other animal cells 15% 1% 29% 16% Figures from ”Expression systems for product and process improvements”, Ronald A. Rader, BioProcess International, June 2008
- 8. Challenges of animal cell culture • Solutions: – Cell Encapsulation – Process Analytical Technology • Need for high cell density cultures • Need for high level of monitoring & control
- 9. Cell Microencapsulation Capsule (Micro-bioreactor) Semi-permeable capsule membrane bioreactor Viable cells Nutrients Shear Critical for the survival stress Wastes of the cells Proliferate Recombinant Protein
- 10. 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
- 11. Development of Novel Microcapsules Aqueous two-phase system – PEG and Dextran Hydrogel membrane Dextran in Polymer • Potential impact • Present work • • Obtaining required characteristics High commercialization possibilities • Cell Encapsulation • Cells suspended within core • Testing new polymers
- 12. Encapsulation of CHO cells Preliminary experiment to optimize cell number in alginate microcapsules Regular shaped and intact microcapsules Empty PLL-alginate microcapsules 0.5x104 cells/ml alginate 1x104 cells/ml alginate Irregular shaped microcapsules Difficulties in jet break up 2x104 cells/ml alginate 2.7x104 cells/ml alginate * 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 *Breguet, V. et al. (2007). Cytotechnology 53: 81-93
- 13. Removal of desired compounds from their associated environments LiB Extraction aides for biotechnological and chemical processes • Alginate hydrogel membrane • Liquid-core • • Hydrophobic material Chitosan, cellulose sulphate etc • Oleic acid, vegetable oils etc • Porous structure Microcapsules Further treatment • Novel approach termed ‘Capsular Perstraction’ Capsular Perstraction Derived from permeation and extraction
- 14. LiB Geldanamycin • Polyketide antibiotic • Ansamycin family • Streptomyces hygroscopicus var geldanus • Received significant attention in 1980’s • Novel antitumor antibiotic Molecular structure Commercial Pelleted growth of geldanamycin geldanamycin (magnification 40X)
- 15. ISPR of Geldanamycin 1.2 1 • Polyketide antibiotic 485 μm 0.8 598 μm • Ansamycin family 751 μm C(t)/C(0) 0.6 • Streptomyces hygroscopicus var geldanus 0.4 • Novel antitumour activity 0.2 • Produced at relatively low conc. 0 • Sensitive to process conditions 0 20 40 60 80 • Liquid-core microcapsules time (min) • Increase productivity Rapid extraction of the antibiotic from degradation environment • 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. Whelehan & Marison (2011). Biotechnology Progress 27: 1068-1077 & Whelehan et al (2012) Journal of Bioscience and Bioengineering (in press)
- 16. Purification of Capsular Geldanamycin + Geldanamycin loaded Capsules (selective removal) Empty capsules Very pure solution for future use for purification Agitate at Geldanamycin, high speed acetonitrile and small quantities of oleic acid Mix with acetonitrile saturated with oleic acid Solution has a higher affinity Removal of oleic acid Acetonitrile removal Low temperature distillation Geldanamycin crystals (purity > 97%) Crystal production
- 17. Application in other fields LiB • Methodology for the treatment of drinking/waste-water • Pharmaceuticals, pesticides and herbicides 120 Sulfamethoxazole 120 Metoprolol Ethylparathion 100 Furosemide 100 Clofibric Acid Methylparathion 80 Carbamazepine 80 % removed Atrazine % removed Warfarin Diclofenac 60 60 2,4 D 40 40 20 20 0 0 0 20 40 60 80 100 120 0 20 40 60 80 100 120 time (min) time (min) • Mechanism to degraded the extracted pollutant Pollutant loaded Pseudomonas capsule Whelehan et al (2010). Water Research 44:2314-24 Wyss et al (2004). Biotech Bioeng 87:734-42
- 18. Determining characteristics of microcapsules • Porosity – HPLC with dextran standards HPLC Chromatogram • Mechanical resistance (strength) – Texture analyzer • Burst Force Before compression After compression
- 19. Data analysis & Management Atomic Force Microscope (AFM) Techniques for capsule Scanning Electron Microscope (Cryo FE-SEM) characterisation Confocal Scanning Laser Light Microscope Microscope (CSLM)
- 20. DUAL STAINING OF LOW GRADE ALGINATE POWDER 633nm laser 488nm laser Polyphosphates- yellow Algin -blue Bar 5mm Combined image
- 21. MONITORING POLYMER STRUCTURAL CHANGES - STAGES Heating/cooling Warm stage (CO2/ N2) Heating/cooling (Peltier) Micro-tensile (Deben UK) Shearing
- 22. Rheology of slow-induced alginate gel 800 700 600 500 G' (Pa) 400 300 200 100 0 0 20 40 60 80 100 120 Time (sec)
- 23. Alginate characterisation Poly- phosphate crystals 500 nm Alginate powder Stored at 55% RH
- 24. Confocal Microscopy Alginate-Poly-L-Lysine-Alginate microcapsules 60 Membrane coat thickness (μm) 40 thickness 20 39± 2μm 0 0 90 180 270 360 position (deg)
- 25. ATOMIC FORCE MICROSCOPY • “Feels” surfaces • Z-resolution ~1 angstrom • Surface topography • Force measurements • Viscoelastic properties
- 26. ATOMIC FORCE MICROSCOPY Chitosan 7 nm spacing Alginate molecules on capsule surface
- 27. AFM - Aqueous-core microcapsules PRE-LIQUEFACTION Height Phase POST-LIQUEFACTION Height Phase
- 28. Post-Liquefaction – Surface topography 208 um
- 29. Characterisation of Encapsulated CHO Cells … 4 × 105 CFU encapsulation
- 30. Are there cells protruding on the capsules surface? Day 0 Day 3 Asylum MFP-3D Day 4 Analysis Mode No cell visible on micro-capsule surface
- 31. Are the cells embedded in the capsule core? Asylum MFP-3D polymer CHO 320 Analysis Mode AFM illustrates cell- polymer interaction within the capsule core
- 32. Encapsulated LIVE CHO DP12
- 33. SINGLE METABOLISING CHO 320
- 34. CHO 320 Encapsulation LIVE/DEAD analysis x10 2 × 104 CFU 4 × 105 CFU 5 × 106 CFU x20
- 35. Encapsulation of CHO 320 Confocal Microscopy … 4 h after CHO cell … 3 × 106 CFU encapsulation encapsulation
- 36. Storage of encapsulated CHO 320 Metabolising CHO cells after 4 days
- 39. “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
- 40. Introduction Encapsulation Technology – Opportunity to address all issues Enhanced Drug Solubility • Drugs available in solubilised form • ↑ Bioavailability Enhanced Drug Permeability • Drug passes into bloodstream • Convert injection into oral • ↑ Bioavailability • Small Molecules • Large Molecules Opportunities • Peptides Enhanced Drug Stability • Once Daily Dosing • Controlled/Targeted release • Lower Dose (Polymer coatings) • Less Side Effects • Targeted Colonic delivery • ↑ Bioavailability 40
- 41. Introduction Encapsulation Approaches Beads Capsules Oil Core Oil Droplets Encapsulated in Gelatin Shell Gelatin Matrix API dissolved in oil/surfactant/co-solvent API dissolved in oil core, mixture entrapped as droplets in a gelatin surrounded by gelatin (or other material) matrix (or other material) shell 41
- 42. Introduction 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 42
- 43. Results – Liquid Formulations Celecoxib Liquid Formulations produced using Optimal Liquid Vehicles 25 liquid formulations prepared containing celecoxib dissolved in combinations of oils/surfactants/co-solvents and assessed via in-vitro dissolution testing 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 43
- 44. Results – Liquid Formulations 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 44
- 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 45
- 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 46
- 47. • Encapsulation of bioactives • Functional foods • Global market size ~$75 billion (GBA, 2007) Global functional foods market 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
- 48. 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?
- 49. Feel free to contact me: Prof Ian Marison Executive Director ian.marison@nibrt.ie Visits can be arranged at any time.
- 50. Thank you for your attention! Any questions?
Notes de l'éditeur
- If we look at biologicals compared to chemical drug substances, we can immediately see why there is this additional amount of understanding needed. Here we have an aspirin molecule and a monoclonal antibody. The difference striking. Therefore, complex compounds need an holistic, integrated approach. Furthermore, complex compounds such as mAb needs correct glycosylation - need for animal cell expression system.
- However, in this work we have achieved this by creating droplets of buffers of a defined composition containing water soluble materials which aide the formation and stability of the droplets. The technique is based on aqueous - 2 - phase systems using a PEG rich phase (core) and dextran rich phase (membrane). These materials help increase the viscosity/surfacetension of the core/membrane materials which enables the droplet to maintain its structure, whereby it can be hardened into a capsule. The picture above show two aqueous-core microcapsules produced using the co-extrusion technique as described above Future experiments will focus on trying to obtain consistently, the characteristics required by capsules (mentioned in previous slide) which are required if they are to be applied to a relevant medical or biotechnology production process. Most importantly experiments will involve the encapsulation of animal cells within the core by mixing the cells in the core material before direct extrusion with the shell material through the defined nozzle The droplets (microcapsules) will contain: (1) water soluble drugs or compounds which are extremely unstable and cannot normally be consumed orally. (2) Vitamin supplements (i.e. folates) for oral delivery, which are essential for the development and growth of healthy foetuses but are readily degraded in the gut. (3) Sensitive mammalian cells capable of producing important recombinant antibodies for use in the treatment of specific diseases and/or for producing diagnostic kits for the early detection of certain ailments and (4) adult stem cells to enable a 3-D platform for tissue production. It is anticipated that the controlled encapsulation of cells and compounds within a hydrogel membrane will help overcome the many problems currently facing the application of such materials in biotechnological and medical processes.
- Pirkko’s results: 1.5 % Na-Alginate in MOPS washing buffer pH 7.0 and filtered with 0.2 micro filter Increasing number of CHO320 cells were mixed in 1.5% Na-Alginate for encapsulation to find optimal seeding cell number Polymerization: 100 mM CaCl2 in MOPS buffer pH 7.0 Result: Encapsulation of cells under 2 x 104 cells / ml alginate is producing regular shaped, intact micro capsules. Next step is to culture encapsulated cells to define the effects encapsulation in growth and define new limiting factors in the microenvironment. Also effects of encapsulation in production and product quality and stability are studied
- Initially we performed experiments to see if capsules were capable of rapidly extractinbg geldanamycin from culture environments and from the graph it can be seen that microcapsules were capable of rapidly extracting the drug from culture environments and this shows the use of different sized capsules didn’t affect extraction speed. The second graph shows the affect of capsule addition on geldanamycin production and growth of the streptomyces with squares representing biomass growth and diamonds geldanamycin production. The red lines are capsule addition experiments. It was discovered that the addition of capsules at day 5 prevented them interfering with the cells. From the results it can be seen that microcapsule assisted fermentations resulted in a 30% higher maximum net concentration of geldanamycin compared to the control fermentation due to the removal of the antibiotic from a hostile fermentation environment. More importantly, the immediate in-situ extraction of the antibiotic resulted in the recovered material been stable in the culture environment for over 24 days whilst not affecting the growth of the bacteria. The process also resulted in a selective removal of the antibiotic, which aided downstream processing
- After recovering the capsules from the fermentation using a very simplistic procedure our next goal was too see if we could recover high quantities of the geldanamycin from the capsules at high levels of purity so that the geldanamycin could be crystallized, whilst also maintaining the microcapsule structure so that they could be used for future experiments. We developed a simplistic procedure to enables to achieve this goal.