Lecture 9.pdf

Cell Expansion for Regenerative Medicine BENG0011 – Manufacturing Regenerative Medicines: From Lab Bench to Industry Dr Rana Khalife Lecture 9 Learning outcomes At the end of this lecture, you should: Understand the differences between regenerative medicine bioprocessing and tradi...

Cell Expansion for Regenerative Medicine BENG0011 – Manufacturing Regenerative Medicines: From Lab Bench to Industry Dr Rana Khalife Lecture 9 Learning outcomes At the end of this lecture, you should: Understand the differences between regenerative medicine bioprocessing and traditional biopharmaceutical production Understand the differences between the different cell manufacturing paradigms Be familiar with the different technologies used for cell production Be able to outline the advantages and disadvantages of the technologies Differences of Regenerative Medicines Compared with Traditional Bioprocesses Cell quality – cell forms the basis of the product Suspension / anchorage-dependent cells – E. coli, CHO cells…stem cells Source of cells – Predominantly derived from primary cells…limited expansion potential – Donor to donor variability Aim of Regenerative Medicine Bioprocessing Create scalable and robust manufacturing processes… that will maintain the critical quality attributes (CQAs) of living cell products… whilst minimizing manufacturing costs of these inherently expensive products. Cell Therapy Manufacturing Paradigms Cell Therapy Manufacturing Paradigms Rafiq et al. 2015  Cell expansion Tissue Biopsy/Blood Cell Isolation Primary Culture Passage/sub-culture Secondary Culture Passage/sub-culture Cell Line Single Cell Isolation Successive passaging Immortalisation Clonal Cell Line Senescence Transformed Cell Line Immortalised Cell Line Cell expansion Jerry W. Shay & Woodring E. Wright Nature Reviews Molecular Cell Biology 1, 72-76 Estimated Cell Requirements Clinical Indication Cell Type Dose (cells/dose) Heart disease Bone marrow stem cells 108 Graft vs. host disease Bone marrow stem cells 109 Liver disease Adipose-derived stromal cells 109 Heart Disease 108 cells per dose Est. 500,000 doses per year (assuming 10% market share of 5M patients) (Mason & Dunnill, 2009) Adapted from Simaria et al 2014  Biochemical Engineering: Translating Science to Society Process Product Target Product Profile Critical Quality Attributes: Commercial Translation Safety Purity Identity Potency Process map deconstruction of a candidate cell therapy bioprocess Ratcliffe et al Br Med Bull (2011) 100 (1): 137-155 Current Manufacturing of Cells for RM Therapies 40 years  Scale-Up Technologies for Cell Expansion T25 T75 T150 https://www.abcscientific.com/ http://www.cellingbiosciences.com/  Scale-Up Technologies for Cell Expansion Different modes of cell culture that can be used: Shake culture Stirred systems Perfused systems How do we determine which mode of culture to use? Variables include culture vessel geometries, culture dynamics, control of feed regime Culture systems (1) 1. T-Flasks 2. Multi-layer Flasks (cell factories) 3. Roller bottles  Automation http://www.tapbiosystems.com/tap/cell_culture/CS_video_gallery/videogallery.h tm CellBase CT Fully Automated T-flasks System Manufactured by Sartorius Stedim Biotech Applications: process development and optimization for scale up, production of clinical material for Phase II and Phase III trials, and Ex vivo expansion of cells for commercial manufacture. Functions: Cell counting, feeding, incubation, passaging and harvest. Features: 1. Aseptic processing environment 2. Precise counting and viability measurement of cell suspensions 3. Processing and tracking of multiple individual patient products in parallel 4. Automated microscopy Scale-Up Technologies for Cell Expansion Sort of achievable scale for each tech Scale-Up Technologies for Cell Expansion Sort of achievable scale for each tech Scale-Up Technologies for Cell Expansion  Planar systems (T-flasks etc.) ❑ Advantages Well accepted and well understood method Reproducible and standardised (esp. with automation) Can be completely automated ❑ Disadvantages Static systems which will result in a heterogeneous culture environment (roller bottles provide a ‘better’ mixed system) – mass and energy transfer gradients) Poor surface area-to-volume ratio Unfeasible for indications require large cell lot sizes (109/1012 cells) Difficult to change mode of operation (i.e. perfusion/fed-batch) Planar culture scalability (Rowley et al. 2012) Culture systems (2) 1. Hollow-fibre bioreactors 2. Packed-bed bioreactors Scale-Up Technologies for Cell Expansion Sort of achievable scale for each tech Scale-Up Technologies for Cell Expansion Hollow Fiber Bioreactor Quantum Cell Culture Platform Manufactured by Terumo BCT Automated Applications: Process Scale up and scale out, Immunotherapy (T-Cell Expansion, CAR T-Cell Based Therapy, Dendritic Cell Maturation), Gene Therapy (Viral Vector Production, Viral Transfection and Transduction), and Cell Therapy (Adherent and Suspension Cell Expansion). Functions: Automated Cell Seeding, Reagent Addition, Feeding and Harvest. Features: 1. 3D hollow-fiber bioreactor maximizes surface area-to-volume ratio 2. Bioreactor surface area equivalent to 120 T-175 flasks 3. Management of 10 Quantum systems by one skilled operator 4. Harvesting cells in as few as 20 minutes 5. Integrated incubator, biosafety cabinet and clean room  Perfusion/Packed-bed Reactors ❑ Advantages Online monitoring and control Better scalability than monolayer culture Improved homogeneity ❑ Disadvantages Difficult to sample (destructive sampling) Potentially uneven cell-distribution – Need to maintain homogeneous axial and radial mass transfer to ensure homogenous cell density throughout Cell removal from reactor may prove difficult Can we use a stirred-tank bioreactor? Microcarriers and STRs Why a microcarrier/bioreactor system? Homogenous, High surface area to well-mixed culture volume ratio environment Able to operate under Production heritage different modes of for biologics operation (e.g. batch, fed-batch, perfusion) Scalable, well Online monitoring and characterised system control  Scale-Up Technologies for Cell Expansion Microcarriers: Most regenerative stem cells are reliant upon anchorage-dependent culture Microcarriers provide a solid matrix/substrate for cell attachment in stirred bioreactors  Scale-Up Technologies for Cell Expansion Microcarriers: Most regenerative stem cells are reliant upon anchorage-dependent culture Microcarriers provide a solid matrix/substrate for cell attachment in stirred bioreactors Scale-Up Technologies for Cell Expansion Microcarriers: Most regenerative stem cells are reliant upon anchorage-dependent culture Microcarriers provide a solid matrix/substrate for cell attachment in stirred bioreactors Many different types of microcarrier, made from different materials are available: Polystyrene, glass, acrylamide, DEAE-dextran, collagen. Can have modified surface chemistries (e.g. peptide-enhanced, aminated to enhance cell attachment) Different materials and surface chemistries can influence cell behaviour such as cell attachment, proliferation and extracellular matrix production. Many types of microcarrier are commercially available: Dextran-based (Cytodex, GE Healthcare), collagen-based (CultiSphere, Percell), and polystyrene-based (Solohill range, Pall). They exhibit a range of different properties e.g. porosity, density, optical properties, surface chemistry and presence of animal component Bioreactor cell densities (Rowley et al. 2012) Comparison of harvest volumes (Rowley et al. 2012)  Scale-Up Technologies for Cell Expansion Other microcarrier-based systems: Stirred systems Fluidized bed Packed bed iCELLis World’s first fully-integrated, single-use Bioreactor Manufactured by Pall Corporation Compact fixed-bed, filled with custom macrocarriers 2 Formats: 1. iCELLis nano (1L) for feasibility studies and small- scale production 2. iCELLis 500 (70L) for industrial scale manufacturing (up to 500 m2) Control over: Temperature, pH, Gas Flow Rates (O2, CO2, N2, and Air), Impeller Speed, and DO. Features: 1. Disposable, pre-installed calibrated probes 2. Integrated mixing system for evenly-distributed media circulation and low shear stress 3. Specialized carriers increase productivity and simplify process 4. Unique waterfall media oxygenation for high oxygen transfer 5. Perfusion device to reduce process development time BIOSTAT STR Bioreactor Manufactured by Sartorius Stedim Biotech Single Use Applications: High density cell culture, industrial and academic research, process development and optimization, pilot scale production, monoclonal antibody, recombinant protein and vaccine production, and seed cultivation for large scale. Features: 1. Available in single and twin versions 2. Up to five addition pumps per single-use vessel 3. Integrated 4-gas mixing system for efficient oxygen transfer and CO2 stripping 4. 6 independently controlled gassing lines 5. Fully equipped single-use bag including standard spargers and impellers 6. BIOSTAT® STR Control Tower is equipped with all necessary measurement and control hardware  Xcellerex XDR Cell Culture Bioreactor System Manufactured by GE Healthcare Bio-Sciences Corp Single use bioreactors Automated Working Volumes: 4.5L to 2000L Applications: cultivation of a wide range of cell types and organisms including CHO cells, Vero cells, and MDCK cells, microbial applications including E. coli, Pseudomonas spp., and yeast, process development and scale up. Control over: Temperature, pH, Gas Flow Rates, Impeller Speed, Dissolved Carbon dioxide, and DO. Features: 1. Both GMP and non- GMP configurations 2. Jacketed stainless steel vessel for exacting temperature control 3. Gas mass flow and liquid pump options for highly productive cultures 4. 3 separate subsystems (vessel/frame, I/O cabinet, and X- Station controller) help maximize the configurability 5. X-Station, a stand-alone, mobile control console  BIOSTAT RM Wave-mixed bioreactor Manufactured by Sartorius Stedim Biotech Single Use System Two Rocker sizes: BIOSTAT® RM 20/50 and BIOSTAT® RM 200 Working Volumes: 100 ml to 100 L Applications: Process transfer from shake or T-flask, rapid material supply for pre-clinical trials, production of recombinant proteins, mAbs and vaccines, continuous cultures with reported cell densities of 150 million cells/ml, seed expansion for large bioreactors, and GMP Production Features: 1. Full GMP compliance 2. Can be used for mammalian (suspension or adherent cells on micro-carriers), insect and plant cells 3. Individual control of two bags on the same platform 4. Advanced alarming and safety features for safe cultivation 5. Reduced manual handling by automated sampling function  Challenges for scale up Typically, things fail! Defining culture parameters that are scalable for human cells is difficult – Switch from static planar culture to other perfused/mixed systems – Predicting success is not possible – Too many parameters change Hydrodynamic shear stress Culture reagents (even those that are batch-validated)  Objective of scale-up To overcome the design conditions and operational procedures to ensure that during the translation of scale, the optimal environmental conditions for the bioprocess (as identified at small-scale) are maintained Environmental conditions 1. Nutrient concentrations 2. Product concentrations 3. Growth inhibiting metabolite concentrations 4. Dissolved oxygen concentration 5. Dissolved carbon dioxide concentration 6. pH 7. Temperature 8. ‘Shear sensitivity’  Process Changes Media Downstream Culture processing Environment Product Impact Cell Surface density Chemistry Topology Summary Major challenges still to be addressed for cell manufacture for regenerative therapy products – primary scale of cell production – Expansion of human cells to therapeutic quantities needs to be achieved. Differing manufacturing paradigms with autologous/allogeneic cell therapies Multiple technologies for production of cells – each with advantages and disadvantages Choosing which cell expansion technology is employed is not straightforward, as technology benefits to the cells need to be considered.