GMP Reagents for Regenerative Medicine: Process Research and Development

Transcription

1 GMP Reagents for Regenerative Medicine: Process Research and Development Mohan C Vemuri Thermo Fisher Scientific Director, R&D Stem Cell Biology 7/9/ The world leader in serving science

2 Agenda MSCs for clinical cell therapy Global trends toward serum free manufacturing Increasing yield through scale up technologies CTS platform & advantages Case studies 2

3 Cells for Therapy ESC ipsc Isolate MSC HSC Fibroblasts NSC Keratinocytes Key themes Expertise Compliance Productivity Expand Differentiate Hepatocytes Deplete Endothelial Neuronal Potency Safety Qualify 3

4 4 Human Mesenchymal Stem Cells

5 Therapeutic applications of MSCs Human MSCs have become of interest for clinical application due to: Immunosuppressive attributes Capacity for homing and engraftment Wide-range mesoderm differentiation potential Potential MSC Therapies: Graft versus Host Disease Crohn s Disease Bone Defects/ Genetic Disease HSC Transplantation Cardiac repair 5

6 Cell Therapy Clinical Trials Major Cell Types 4% 2% 3% 13% 31% MSC total (90) Immune cell total (140) BM MNC (38) Adipose SVF (12) Skin cells (7) Cord Blood MNC (9) 47% Bersenev Alexey. Cell therapy clinical trials 201 report. CellTrials blog. February 1, Available: report/ 6

7 7 Global trends toward serum free manufacturing

8 Trends towards serum free culture Supply of FBS is predicted to be unable to support the cell therapy industry if more than one blockbuster therapy hits the market The use of serum carries with it the risk of transmission of adventitious agents Lot to lot variability presents a challenge for manufacturing reproducibility and adds cost from a Quality and lot selection perspective Serum can potentially cause xenogeneic immune response in patients 8

9 The Evolution of Human MSC Medium Classical Human MSC Media Basal media with 10-20% FBS Eliminate need for pre-qualification of reagents Decrease lot-to-lot variability and increase performance Reduced Serum Complete Media MesenPRO RS (reduced-serum + GFs) Eliminate the need for xenogeneic serum Alternative Human MSC Media Basal media with 10% Human Serum or Human Platelet-Rich Plasma Lysate Eliminate need for pre-qualification of reagents Decrease lot-to-lot variability and Increase performance Eliminate the need for serum (1) Serum-Free or (2) Serum-Free and Xeno-Free 9

10 Serum-supplemented Media Pros: General growth promoting properties for many cell types FBS: historically used with many cell types Pooled human serum: used with primary cells (T Cells, etc.) Commonly used, most historical functional data generated with serumcontaining formulations Cons: Undefined (complex mixtures of components) Potential for lot-to-lot variability Risk of contamination by adventitious agents Lot size limitation 10

11 Why Serum-free? Advantages: Defined components and reduced variability Reduce exposure to adventitious agents Serum-free xeno-free animal origin-free (AOF) Advantage Path to 510K GMP Large scale lot production Challenges: Cost-of-goods (COGs) Recombinant proteins/sourcing/function Impact on cell phenotype and function 11

12 Serum-free Alternatives Serum-free: may contain native proteins from animal sources Xeno-free: may contain native proteins from human sources only Platelet lysate from pooled donors, functional, but undefined Animal origin-free (AOF): No native proteins from animal sources (including human) May contain recombinant proteins/growth factors/etc. Chemically-defined: No proteins or undefined components (proteins, hydrolysates) 12

13 Gene Expression Analysis to Facilitate Media Design Osteogenic Basal Osteo Adipogenic Basal Adipo Chondrogenic Basal Chondro Analysis of active pathways in human MSCs suggests that PDGF, TGFβ and FGF are important signaling pathways for hmsc proliferation and differentiation 13 Ng et al., Blood, 15 July 2008, Vol. 112, No. 2, pp

14 Advanced Human MSC Media Systems MesenPRO RS - Reduced (2%) Serum-Containing Medium StemPro MSC SFM - Serum-Free MSC Medium StemPro MSC SFM XenoFree - Serum-Free/Xeno-Free Medium A complete line of cgmp-manufactured advanced MSC media Table 1. MSC culture product selection guide Culture System Basal Media Comple te Media Supplements Cell / Tissue Species Cell / Tissue Source Subs trates Passaging Serum- Classical Media Reducedserum DMEM (low glucose) / alpha MEM MSC-Qualified FBS MesenPRO RS Human, mouse, sheep, pig, canine, horse, rat Human, mouse, sheep, pig Serum-free StemPro MSC SFM Human Serum-free / Xeno-free StemPro MSC SFM XenoFree Human Bone Marrow Adipose Cord Blood Umbilical Cord Placenta Bone Marrow Adipose Umbilical Cord Bone Marrow Adipose Umbilical Cord Hair Follicle Bone Marrow Adipose Umbilical Cord Cord Blood Pericytes Fibroblasts CELLstart, Fibronectin, Attachment Factor CELLstart, Coating Matrix, Fibronectin TryPLE, Trypsin, StemPro Accutase TryPLE, Trypsin, StemPro Accutase TrypLE TrypLE 14 For research use only. Not intended for human or animal therapeutic or diagnostic use.

15 Reduced Serum MSC Culture System MesenPRO RS Medium Osteoblasts (Alizarin Red S) Reduced (2%) serum with consistency and variability Eliminates need to pre-qualifying FBS Utilizes product-specific human MSC quality control assay Maintains multipotent MSC phenotype Gene expression profiles matches classical medium Adipocytes (Oil Red O) Chondrocytes (Alcian Blue) NOTE: Recommended seed density = 5x10 3 cells/cm 2 15 For research use only. Not intended for human or animal therapeutic or diagnostic use.

16 Serum-Free MSC Culture: StemPro MSC SFM CTS Medium A Relative Flourescence Units (RFU) P = PDGF-BB B = bfgf T = TGFβ1 SCM D+PBT D D+P D+B D+T D+PB D+PT D+BT Growth Factor Supplementation C Net Population Doublings Set-Up Passage SFM SCM NOTE: Recommended seed density = 1x10 4 cells/cm 2 B Optimized mix of recombinant growth factors provides an enhanced synergistic effect on human BM-MSC proliferation. Optimized formulation provides ability for continual propagation. a b More spindle-shaped morphology, supporting high density cell growth. System utilizes a humanized substrate (CELLstart ) and a recombinant trypsin alternative (TrypLE ) 16 Chase et al., Stem Cell Research & Therapy 2010, 1:8. For research use only. Not intended for human or animal therapeutic or diagnostic use.

17 Xeno Free MSC Culture: StemPro MSC SFM XF Medium StemPro MSC SFM XenoFree DMEM + 10% MSC-Qualified FBS StemPro MSC SFM XenoFree: Net Population Doublings (Human BM-MSC) Expansion and Differentiation Passage 1 Passage 9 Input cells = passage 5 human Bone Marrow MSCs (4-donor pool) Net Population Doublings Set-up DMEM + 10% MSC-Qualified FBS Passage StemPro MSC SFM XenoFree StemPro MSC SFM XenoFree: Doubling Time (Human BM-MSC) Recommended seed density = 5-7x10 3 cells/cm Adipocyte (Oil Red O) Chondrocyte (Alcian Blue) Osteoblast (Alkaline Phosphatase) Doubling Time (Hours) Passage DMEM + 10% MSC-Qualified FBS StemPro MSC SFM XenoFree Passage 5 Multi-lineage Mesoderm Differentiation with StemPro Differentiation Regents Multi-passage expansion of human bone marrow-derived MSCs 17 For research use only. Not intended for human or animal therapeutic or diagnostic use.

18 MSC Media System Summary Media System Catalog Number Additional Reagents StemPro MSC SFM XF A10675 CELLstart CTS, GlutaMAX I CTS, TrypLE Select CTS, gentamicin (optional) StemPro MSC SFM CTS A10332 CELLstart CTS, GlutaMAX I CTS, TrypLE Select CTS, gentamicin (optional) MesenPRO RS GlutaMAX I CTS, TrypLE Select CTS, gentamicin (optional) DMEM low glucose + 10% MSC Qualified FBS GlutaMAX I CTS, TrypLE Select CTS, gentamicin (optional) StemPro Osteogenesis Kit A10072 gentamicin (optional) StemPro Adipogenesis Kit A10070 gentamicin (optional) StemPro Chondrogenesis Kit A10071 gentamicin (optional) 18

19 19 Increasing yield through scale up technologies

20 Process considerations and control Bioprocess Connect biology and engineering Establish process, manufacturing, scale and drive COGs What are the critical considerations? Quality of supporting materials and reagents Process choices Development & regulatory challenges Biology Bioprocess En Scalable & robust processes should be selected with thoughts of commercialization to avoid costly change control in the future 20

21 Lot-size Challenges Planar Cultures Packed Bed / Suspension Cultures Product Doses 21 Rowley et al., Bioprocess International, March 2012

22 Increasing Yield to Meet Demand Viable Cell Density (VC/ml) 2.5e+7 2.0e+7 1.5e+7 1.0e+7 5.0e+6 Control VCD Optimized VCD Control Titer Optimized Titer IgG Titer (mg/l) Monoclonal Antibody Production Substantial increase in yield over the last 20 years Time (Days) Control Process Optimized Process Allogeneic Cellular Therapy Similar progression needed for CT Doses per Lot

23 Culture Media Design Strategy Mix Empirical and Focused Methods: Checkpoint DOE Metabolite analysis Cytokine/growth factor pathway Characterization checkpoints Gene expression analysis High throughput studies Checkpoint 9 Fold Increase Cell Growth Medium DOE Medium Variant Checkpoint 23

24 Scalability Cell Density Cells (cells/ml) / ml A 2.5E E E E E E+00 BM MSC ASC 100ml spinner flask (n=2) Cell Density (cells/ml) B 1L bioreactor (n=2) Time (days) StemPro MSC SFM XenoFree Solohill plastic microcarriers coated with CELLStart CTS Incorporation of partial medium exchanges Yield of MSCs (~ cells/ml) A completely xeno-free microcarrier-based culture system was successfully implemented for the expansion of human MSC. A dos Santos et al., Tissue Eng. Part C,

25 Medium Optimization: Nutrient Feed Development Used metabolite and spent media data from previous bioreactor run to design a nutrient feed. Tested fed-batch approach based on calculated consumption rates Cell Density (cells/ml) 2.5E E E E E+04 Fed batch 25% 2 days 25% 25% Exchange daily every other day 25% Exchange daily 0.0E Time (days) Fed-batch approach showed comparability to partial medium exchange 2014 Jan 14. doi: /bit [Epub ahead of print] 25

26 MSC Colony Number Greater Under Hypoxic Conditions (P2 culture) Grown under hypoxic conditions. Grown under normal oxygen conditions. Greater MSC colony formation following expansion under hypoxic environment vs. normal oxygen Greater MSC colony formation following isolation and expansion under hypoxic environment vs. normal oxygen 26

27 27 CTS platform & advantages

28 CTS Brand Translational tools FDA DMF or 510(k) ISO & GMP manufacturing Animal origin-free or xeno-free Certificates of Origin Certificates of Analysis Adventitious agent testing Sterility testing Mycoplasma testing Endotoxin testing 28

29 Cell Therapy Systems (CTS) Regulatory Considerations Customers in Cell Therapy space need access to tools and reagents that allow them to transition from research applications into clinical applications Current regulatory guidelines are not black-and-white and are evolving as the field emerges The goal of the CTS product line provide qualifying reagents as developers prepare to move their concepts into the clinic What CTS offers High quality materials Unified documentation for CTS products Certificate of Analysis > Certificate of Origin Animal origin designation> Adventitious agent testing Product reserves (lot specific) > Raw material alerts Drug/Device Master File (FDA) for relevant products Technical, regulatory, and web-based support Dedicated support team Media, Buffers, Growth Factors, Enzymes, Supplements, Devices 29 Life Technologies Proprietary & Confidential

30 Process Check for cgmp Reagents Reagent & media labeling 510K cleared media products, Drug Master File Animal Origin Free, Xeno-Free, IVD For manufacturing of cell-based products (CTS) Quality systems under which reagents are manufactured (21 CFR Part 820) ISO 13485:2003 Certification Raw Material Sourcing Audits Paper trail/validated vendors Retest Documentation to support traceability, quality, and intended use 30

31 Thermo Fisher Cell Therapy Provider of stem cell and cell engineering tools for development Enabling rapid progress from cell to market Provider of workflow solutions for isolation and expansion of cells Custom solutions and scalable manufacturing from clinical to commercial stage Used in a broad range of cell therapy applications where consistency, quality and safety are critical Consultative partner providing deep domain knowledge and experience Scalable and reliable solutions for cell therapy research and production 31

32 Immune Cells for Therapy Four major types :CIK, T cells, Dendritic cells and Natural killer (NK) cells Cytokine-induced killer (CIK) cell: Cytokine-induced killer (CIK) cells are CD3-and CD56-positive, non-major histocompatibility complex (MHC)-restricted. When reintroduced back to patients after autologous stem cell transplantation, CIK cells may recognize and kill tumor cells associated with minimal residual disease (MRD). (21 clinical trials*) Natural killer (NK) cell: A population of activated, immortalized, interleukin-2 (IL-2)-dependent, cytotoxic natural killer (NK) cells with potential antitumor activity. (273 clinical trials* ) Dendritic cell (DC): Immune cells that process antigen material and present it on the surface to other cells of the immune system. They act as messengers between the innate and adaptive immunity.(403 clinical trials* ) T Cells : T cells or T lymphocytes belong to a group of white blood cells known as lymphocytes, and play a central role in cell-mediated immunity (5427 clinical trials* ) *Clinical trial data ( US centric Pre-Clinical and Cinical studies with CIK cells 32

33 T Cell Workflow for Immunotherapy 33

34 Serum-free Process for T Cell Expansion CD3/CD28 activated T Cells are typically expanded in media supplemented with human serum. In order to make a commercial-scale process possible, a GMP, regulatory-compliant, scalable substitute is needed. 34

35 Serum-free Process for T Cell Expansion DOE media variant performance Medium 10 Medium 9 Medium 8 Medium 7 Medium 6 Medium 5 Medium 4 Medium 3 Medium Normalized T cell growth (seven donors) 35

36 Serum-free Process for T Cell Expansion A GMP xeno-free serum replacement supplement was developed Growth Kinetics from One Representative Donor Control + 5% HS Control + 10% SRS-XF Control + 5% SRS-XF OpT 2% HS N=4 OpT 10% SRS N=4 OpT 5% SRS N=4 OpT 2.5% SRS N=4 OpT N=4 Control + 2% SRS-XF Control (no additions) T cells expanded in control medium supplemented with human serum or SRS-XF show similar growth kinetics and total fold expansion after 2 weeks in culture. OpT 2% HS N=4 OpT 10% SRS N=4 OpT 5% SRS N=4 OpT 2.5% SRS N=4 OpT N=4 2-5% SRS-XF support expansion of CD4 and CD8 T cell subsets when expanded in OpTmizer CTS 36

37 CTS Immune Cell SR Benefits Intracellular IFN production 5% SRS-XF 5% AB serum Security of Supply Qualified suppliers Multiple GMP sites 42 % 25% Superior performance Traceable Superior Intracellular IFN production COO, COA, Donor Testing, DMF For in vitro diagnostic use Regulatory compliant & commercial path Saves time and cost Eliminate need to qualify serum Similar performance with reduced level Flexible Immune Cell Qualified Bead based QC Assay Scalable process 37 Can replace human serum or FBS and be used with different media

38 T Cell Workflow for Immunotherapy Dynabeads CD3/CD28 CTS Blood Collection DPBS CTS Infusion Cells Activated Cell Expansion Bead Removal and Formulation DynaMag CTS AIM V CTS OpTmizer CTS IL-2, IL-7 CTS, IL-4 CTS DynaMag CTS 38

39 CIK Clinical Immunotherapy Workflow Peripheral blood Collection Harvest, Rinse, Verify CIK & Infuse into Patient What s needed for CIK cultures? PBMNC isolation Cells stimulated with IFN-γ, CD3 antibody Patient Expansion in culture bag Cultured for days Medium Culture bag/culture vessel IL-2, IFN- γ, CD3 Human AB serum CD3, CD4, CD8, CD56 for FACS Ficoll-Paque, centrifuge tubes (fresh PBMNC prep) 39

40 BioProcess Optimization for CTL Production Wilson-Wolf Vera et al ; 2010 J Immunotherapy 40

41 Input-Output Control Design for DC Cultures CD-206+ (DC) CD-86+ (DC) CD-80+ (DC) CD-14+ (Monocyte) *In order to be a DC all three receptors must be expressed 41

42 Media Systems for Immune Cells Cell type Tx Time CIK 3-4 wk RPMI L Retronectin (for Takara SOP), CD3 ab Media Vol/tx Substrate Cytokines/Abs - IL-2, IFNg - CD3, CD4, CD8 CD56, NK 3-4 wk RPMI L Feeder cells - IL-2, IL-15 - CD3, CD4, CD8, CD56 DC 10 d RPMI 1640 or AIM-V Harvest Cell No. 3L N/A - GM-CSF, IL-4, TNFa 2e CD1a, CD40, CD80, CD83, CD86, HLA-DR All All Release assay - FACS - Possible functional assay - LDH assay -FACS - Possible functional assay -FACS - Possible functional assay T cell (multiple subtypes incl Treg) 10 d to 4 wk RPMI 1640, AIM-V or OpTmizer 3-10 L CD3 ab - IL-2 - CD3, CD4, CD8, CD25, CD28(?), CD45, CD56 3e FACS - Possible functional assay 42

43 Summary: T Cells/CIK A GMP compatible serum free and xenofree culture media and a bioprocess optimization for human T Cells was developed efficiently to meet autologus therapy demands of T cells. CIK cells can be cultured efficiently for adoptive immunotherapy needs and holds a greater promise for future. Media and process development for scalable approaches can take a great idea and transform it into a cost effective and efficient therapy. 43

44 State-of-the-art Manufacturing Facilities Cramlington, UK Single-Use technologies Class 10,000/ISO 7 clean room Newport, UK ISO 9001:2008 Paisley, Scotland Cell culture media, reagents ISO GMP 21 CFR 820 Roskilde, Denmark ISO 13485:2003 ISO 9001:2008 FDA Registered Facility Logan, Utah Single-Use technologies Class 10,000/ISO 7 clean room Warrington, UK Analytics kits Suzhou, China ISO ISO 9001 ISO Grand Island, NY Cell culture media, reagents Sera ISO GMP 21 CFR 820 Bedford, MA Chromatography resins ISO Rochester and Fairport ISO 9001:1994 FDA Registered Facility Miami, OK ISO 9001:2008 Oslo & Lillestrøm, Norway Dynabeads Auckland and Christchurch, New Zealand Newcastle, Australia Sera Protein products Naarden, The Netherlands GMP 21 CFR 820 Affinity ligands 44

45 Acknowledgments Andy Campbell Yuan Wen Shayne Boucher Sandy Kuligowski Eric Roos Angel Rohena-Varela Melanie Andolina Karoline Schjetne Brian Newsom Mohan Vemuri Mark Powers Tanja Aarvak Stephen Gorfien Scott Jacobia Grethe Økern Instituto Superior Tecnico Cláudia Lobato da Silva Francisco dos Santos Pedro Z. Andrade Manuel M. Abecasis Joaquim M.S. Cabral Thermo Fisher Scientific Inc. All rights reserved. Solohill is a registered trademark of Solohill Engineering, Inc. 45