Pluripotent Stem Cells for Drug Development and Therapy: Bioprocess Challenges

Transcription

1 Pluripotent Stem Cells for Drug Development and Therapy: Bioprocess Challenges Paula Alves Animal Cell Technology Unit ibet Instituto de Biologia Experimental e Tecnológica

2 Human Stem Cells applications 2 Pluripotent Stem Cells Multipotent Stem Cells Adult Stem Cells Somatic cell Blastocyst Drug Screening & in vitro toxicology Disease Modelling Regenerative Medicine & Tissue Engineering

3 CELL-BASED PRODUCTS: INCREASED COMPLEXITY 3 Courtesy: G. Russotti, Celgene Cellular Therapeutics Small Molecule (e.g. aspirin) Recombinant protein (e.g. human Growth Hormone) Monoclonal Antibody (e.g. IgG1) Differences in mass by ~ 1x10 12 in surface area by ~ 5x10 6

4 Environmental factors affecting hpsc s fate 4 Serra et al., Trends in Biotechnology 2012

5 MOTIVATION 5 Typically cells/patient CHALLENGES: STEM CELL MANUFACTURING PLATFORMS (compliant with GMP) & PRODUCT CHARACTERIZATION (QC/QA) Serra et al. (2012), Trends in Biotechnology

6 NEEDS 6 Cryopreservation Formulation; Storage Transportation THE PRODUCT Quantity THE BIOMANUFACTURING PLATFORM CELL EXPANSION CELL DIFFERENTIATION CELL PURIFICATION Quality Purity CELL CHARACTERIZATION

7 CRITERIA FOR PRODUCTION OF hipsc/hesc QUALITY QUANTITY High Cell Viability Pluripotent Phenotype (no spontaneous differentiation) Normal Karyotype Pluripotent Potential 7 Clinically-relevant cell numbers ( cells)

8 Production of Human Stem Cells 8 Scalability Scale-out ````` Automation Reproducibility

9 9 Cell Monolayer Cultures Scale-up Cell Monolayers Limited Scale-up Useful for process development Roller-Bottles Cell Factories

10 Production of Human Stem Cells 10 Scalability Scale-up ````` Automation Reproducibility Scalability Monitor & Control Photo: courtesy A.Amanullah

11 Strategies to move Anchorage dependent cells into Suspension

12 Perfusion Stirred-tank Bioreactors: monitor & control SCALE: 50 ml 30 L

13 Long term cultures to feed high throughput or chips but also for repeated dose Tox Feeding high-throughput systems: High content screening Functional assays Toxicological assays Drug screening & drug-drug interactions Feeding organ-on-a-chip: Tight control on culture parameters 13

14 WHAT BIOREACTOR? DOES IT MATTER? 14 Mobius CellReady PBS Bioreactor PadReactor DASGIP ambr Bioreactor Wave Bioreactor Biostat

15 IMPACT OF THE BIOREACTOR TYPE & AGITATION STIRRED TANK BIOREACTOR DAPI/ αmhc-gfp / Collagen type I WAVE BIOREACTOR 100 μm WAVE-INDUCED AGITATION ACCELERATES DIFFERENTIATION OF ips CELLS INTO CMS HIGHER DEPOSITION OF COLLAGEN TYPE I 15

16 Impact of Bioreaction Parameters: O 2 & Agitation Cardiomyocyte differentiation yield 16 Cardiomyocyte Differentiation Yield = No. of CMs No. of ips cells >1000-FOLD % O 2 CONTINUOUS 12 4% O 2 CONTINUOUS 4% O 2 INTERMITTENT 4% O 2 WAVE IMPROVEMENT 2.3 x10 9 CMs per 1L bioreactor run

17 17 BIOPROCESS DESIGN FOR STEM CELL MANUFACTURING

18 18 BIOPROCESSING OF HUMAN PLURIPOTENT STEM CELLS & DERIVATIVES Neural hescs & hipsc Simão D et al 2015, Tissue Eng Part A Terrasso AP et al 2015, J Biotechnol Simão D et al 2016, Gene Therapy Sá JV et al 2017, Nuerochem Res. Simão D et al 2016, Sci Rep Terrasso AP et al 2017, J PharmToxicol Meth Cardiac Serra et al 2010, J Biotech; Serra/Correia et al 2011, PLoS ONE; Silva et al 2015, Stem Cells Transl Med; Abecasis et al 2017, J Biotech Cunha et al 2017, J Membrane Science Serra et al 2012, Trends in Biotech; Correia et al 2014, Stem Cell Rev Rep; Gomes-Alves et al 2014, Proteomics; Gomes-Allves et al, 2015, Transl Research; Correia et al 2016, Stem Cell Transl Med; Correia et al 2017, Sci Rep; Correia et al 2018, Biotecnology & Bioengineering Tostões RM et al 2011, Biotech Bioeng Leite SB et al 2011, Toxicol In Vitro Tostões RM 2012, Hepathology Leite SB et al 2012, Toxicol Sci. Rebelo SP et al 2015, Arch Toxicol. Rebelo SP et al 2017, J Tissue Eng Regen Med.. Hepatic

19 19 BIOPROCESSING OF HUMAN PLURIPOTENT STEM CELLS & DERIVATIVES hescs & hipsc Cardiac

20 - TITLE OF SLIDE CHALLENGES TO THE USE OF PLURIPOTENT STEM CELLS FROM TO PLURIPOTENT STEM CELLS (ESCS/IPSCS) THE PROMISE POWERFUL UNLIMITED SOURCE OF CMS FOR: Cardiac cell therapy; In vitro cardiac disease modeling; Patient-specific cardiotoxicity drug screening; 20 FUNCTIONAL CARDIOMYOCYTES (CMS) THE WEAKNESS Low Differentiation Yields / Purity Reduced Scalability & Reproducibility Immature phenotype

21 AIMS DEVELOP NOVEL APPROACHES FOR ROBUST GENERATION OF RELEVANT NUMBERS OF hpsc- CMS WITH HIGH PURITY AND IMPROVED MATURITY FOR CLINICAL AND PRE-CLINICAL APPLICATION hpsc EXPANSION CARDIOMYOCYTE DIFFERENTIATION CARDIOMYOCYTE MATURATION MORPHOLOGY STRUCTURE CELL CHARACTERIZATION METABOLOMICS FLUXOMICS TRANSCRIPTOMICS GENE AND PROTEIN EXPRESSION ULTRASTRUCTURE FUNCTIONALITY 21

22 EXPANSION OF hescs IN BIOREACTORS USING MICROCARRIER TECHNOLOGY Perfusion system 1. Cell retention Custom probe with ceramic filter 2. Precise medium addition/withdrawal Gravimetric control 3. Automated Computer controlled algorithm DAPI/Oct-4 Robust bioprocess for expansion of hesc (without differentiation); Well-defined culture conditions; 15-fold improvement in cell expansion yields when compared to 2D cultures 22 Serra M. et al (2010) J. Biotechnol; Serra M. et al (2011), Plos ONE; Silva MM et al. (2015), Stem Cells Trans Med 2015

23 23 PREVIOUS WORK CULTURE HUMAN (PLURIPOTENT STEM) CELLS AS 3D AGGREGATES IN STIRRED CULTURE SYSTEMS Aggregate size impacts on hpsc growth and quality 3x10 6 cell/ml 6-fold cell expansion in 7 days

24 AIM: EXPANSION OF HPSC Development of a protocol for the CONTINUOUS EXPANSION of hipsc as 3D aggregates in stirred-tank bioreactors Robust Scalable Integrated GMP-compatible Cost-effective BIOPROCESS INTENSIFICATION I. Expansion of hipsc using Bioreactors in perfusion II. Dissociation of 3D cell aggregates III. Sequential Passages In process analytics screen for characterization of hpsc quality attributes 24 Abecasis B et al. (2017), J Biotechnol

25 STRATEGY Maximize Volumetric Cell Concentration and Expansion Factors DasGip Parallel Bioreactor Systems Working volume 200 ml Culture Medium Cellartis DEF-CS TM Xeno-Free Medium Impact of 2 Critical Process Parameters: Dissolved Oxygen: 20% O 2 versus 4% O 2 Perfusion Rate: Low versus High I. Expansion of hipsc using Bioreactors in perfusion II. Dissociation of 3D cell aggregates III. Sequential Passages 25 Abecasis B et al. (2017), J Biotechnol

26 Day 2 Day 2 /GLC Total cell concentration (x10 6 cell/ml) Day 4 Day 4 Day 0 (5h) Day 0 (5h) C BR_20%O 2 2 BR_4%O 2 2 BR_4% IMPACT OF DISSOLVED OXYGEN ON hipsc EXPANSION BR_ Perf LOW versus BR_ Perf LOW 5 4 Cell Growth Profile Aggregate Size and Cell Viability LIVE DEAD µm 100 µm Culture Time (day) Lower DO resulted in higher volumetric cell concentrations and expansion factor as well as aggregates with higher diameters; In both conditions, cells show high viability and glycolytic metabolism 26 Abecasis B et al. (2017), J Biotechnol D D BR_20%O 2 2 BR_4%O 2 2 BR_4%O Y LAC _20%O2 R_4%O2

27 (mmol/l) Total cell concentration (x10 6 cell/ml) IMPACT OF PERFUSION RATE ON hipsc EXPANSION BR_ Perf LOW versus BR_ Perf LOW versus BR_ Lactate Concentration C inhibitory Maximum Cell Concentration fold improvement Culture time (day) BR_20%O2 BR_4%O2 Perfusion Rate was rationally increased in order to maintain lactate concentration at noninhibitory levels, resulting in improved hipsc expansion factor; 2 0 BR_4%O2POPT Abecasis B et al. (2017), J Biotechnol 27

28 IMPACT OF PERFUSION RATE ON hipsc EXPANSION 28 Abecasis B et al. (2017), J Biotechnol (L) BR_ Perf LOW versus BR_ Perf LOW versus BR_ For a Manufacturing Scale of cells: Bioreactor Volume BR_20%O2 BR_4%O2 BR_4%O2Perf Downsize bioreactor volume to 2.1 L 2.1 reduction of the manufacturing footprint and Cost of Goods.

29 Expansion factor of viable cells CONTINUOUS EXPANSION OF hipsc AGGREGATES IN BIOREACTORS 3 sequential passages 10 4 Expansion Factor P1 (4 days) P2 (3 days) P3 (4 days) Culture time (day) hipsc showed reproducible cell growth during 3 sequential passages - total cell expansion factor of 1100 in viable cell was achieved in 11 days; Abecasis B et al. (2017), J Biotechnol 29

30 Percentage of positive cells CONTINUOUS EXPANSION OF hipsc AGGREGATES IN BIOREACTORS Pluripotent Phenotype Day 0 Passage 1 Passage 2 Passage 3 Karyotyping 20 0 Oct4 SSEA-4 Sox17 SSEA-1 Pluripotent Potential ECTODERM ENDODERM MESODERM hipsc maintained Pluripotent Phenotype and Potential and a stable Karyotype 30 Abecasis B et al. (2017), J Biotechnol

31 AIMS HPSC EXPANSION CARDIOMYOCYTE DIFFERENTIATION CARDIOMYOCYTE MATURATION MORPHOLOGY STRUCTURE CELL CHARACTERIZATION METABOLOMICS FLUXOMICS TRANSCRIPTOMICS GENE AND PROTEIN EXPRESSION ULTRASTRUCTURE FUNCTIONALITY 31

32 MATURATION OF hpsc-cardiomyoctes: BACKGROUND hpsc-cms are still highly immature showing metabolic, structural and functional characteristics that more closely resemble fetal CMs than adult CMs FETAL-LIKE CM ADULT-LIKE CM Human PSC-CMs Adult human CMs 32 Adapted from White et al.2016, Food Chem. Toxicol

33 - TITLE OF SLIDE MATURATION OF hpsc-cardiomyoctes: WORKING HYPOTHESIS During heart development CM maturation has been associated with a transition from an embryonic-like glycolytic metabolism to an adult-like oxidative metabolism FETAL 18% 13% 25% 5% 5% 44% 41% 49% 12% ADULT 7% 1% 80% Glycolysis Fatty acid β-oxidation Lactate Oxidation Glucose Oxidation Adapted from Lopaschuk et al. 2010, J. Cardiov Pharmacol QUESTION: Can we improve structural and functional maturation of hpsc-cm by modulating their metabolism?

34 hpsc - TITLE OF SLIDE hpsc-cm (fetal-like CM) hpsc-cm (adult-like CM) EXPERIMENTAL DESIGN CM DIFFERENTIATION d0 d10 d20 Glc-M RPMI w/ Glucose GalFA-M RPMI w/o Glucose w/fatty acids and Galactose [1], [2] CELL CHARACTERIZATION FLUXOMICS METABOLOMICS TRANSCRIPTOMICS MORPHOLOGY STRUCTURE ULTRASTRUCTURE FUNCTIONALITY 34 Correia C. et al Sci Rep [1] Aguer et al. 2011, PLoS One; [2] Marroquin et al. 2007, Toxicol. Sci.

35 METABOLOMIC ANALYSIS Glc-M d20 GalFA-M d20 FA and Gal are oxidatively metabolized via TCA cycle and OXPHOS for ATP generation; 2.7-fold improvement ATP production; Glycolytic genes were down-regulated and most genes related to TCA cycle, OXPHOS and FA metabolism were up-regulated. 35 Correia C. et al Sci Rep IMPROVED METABOLIC MATURATION IN GalFA-M

36 Unstructural analysis Structural analysis Cell morphology analysis Circularity Lenght-to-Width Ratio Area ( m 2 ) STRUCTURAL ANALYSIS Glc-M d20 GalFA-M d *** ns *** ns *** *** *** *** *** GLCM GLCM GFAM Glc-M d20 GalFA-M d d0 d d0 d0d20d20 IMPROVED STRUCTURAL MATURATION IN GalFA-M More elongated morphologies; More organized sarcomeric structures; Higher myofibril density and sarcomere alignment. 36 Correia C. et al Sci Rep

37 FUNCTIONAL ANALYSES Calcium transient In collaboration with Dr Ibrahim Domian GalFAM Contractility Action Potential 37 Correia C. et al Sci Rep Faster calcium-transient kinetics; IMPROVED FUNCTIONAL MATURATION IN GalFA-M Higher percentage of shortening and contractile force; Enhanced AP durations and upstroke velocities. 37

38 TAKE HOME MESSAGE STEM CELL MANUFACTURING CONTROL OF CPP MAXIMIZE STEM/PROGENITOR CELL EXPANSION ENHANCED CELL QUALITY LOW PROCESS CYCLE TIMES HIGH CELL VOLUMETRIC PRODUCTIVITY CONTINOUS EXPANSION SMALLER MANUFACTURING FOOTPRINT LOWER COG AND BIOREACTOR PERFUSION SYSTEMS EASILY CHANGE CARBON SOURCES 3D AGREGATE CULTURE CONTROL CELL FATE (DIFFERENTIATION AND MATURATION) FACILITATED CELL DIFERENTIATION FLEXIBILITY ADVANCED 3D CELL-BASED MODELS MORE MATURATED PHENOTYPES HIGH CELL QUANTITY AND QUALITY FOR: REGENERATIVE MEDICINE DEVELOPMENTAL BIOLOGY 38 DISEASE MODELING PHARMACOLOGICAL STUDIES

39 Animal Cell Technology Unit Cláudia Correia Univ Skövde & AstraZeneca) Alexey Koshkin Ana Teixeira ETH Zürich) Patrícia Duarte Patrícia Alves Maria João Sebastião Inês Isidro Margarida Serra Manuel Carrondo Collaborations Ibrahim Domian & Dongjhan Hu Harvard Medical School, Boston, USA David Elliott Murdoch Childrens Research Institute, Australia Anders Aspergen TBE- CELLARTIS AB, Gothenburg, Sweden 39 Animal Cell Technology Unit Funding ACKNOWLEDGEMENTS

40 Thank you