Table of Contents

Cover

Title Page

List of Contributors

Preface

Chapter 1: Overview of the Cell Therapy Field

1.1 The Context of Cell Therapies and Their Manufacturing Challenges
1.2 The Cell Therapy Landscape
1.3 Operations in Cell Therapy Manufacture
1.4 Upstream Processing of Cellular Therapies
1.5 Downstream Processing of Cellular Therapies
1.6 Formulation, Fill and Finish of Cellular Therapies
1.7 Administration of the Cell Therapy to a Patient
1.8 Cell Therapy Manufacturing Facilities of the Future
1.9 Conclusion
References

Chapter 2: Structured Methodology for Process Development in Scalable Stirred Tank Bioreactors Platforms

2.1 Introduction
2.2 Understanding the Engineering of the Stirred Tank Bioreactors
2.3 Understanding the Biology of the Cells in Stirred Tank Bioreactors (STB)
2.4 Process Development of Adherent Cells in STB Platforms
2.5 Future Directions
References

Chapter 3: The Effect of Scale-up on Cell Phenotype: Comparability Testing to Optimize Bioreactor Usage and Manufacturing Strategies

3.1 Introduction
3.2 Challenges in Cell Product Development
3.3 Stem Cell Characterization
3.4 Next-generation Stem Cell Development
References

Chapter 4: The Scale-up of Human Mesenchymal Stem Cell Expansion and Recovery

4.1 Introduction
4.2 Scale-up or Scale-out
4.3 Understanding the Small Scale
4.4 Microcarrier Screening
4.5 Spinner Flask Culture
4.6 Large-scale Expansion in Conventional Stirred Tank Bioreactors
4.7 Cell Recovery from Microcarriers
4.8 Conclusions
References

Chapter 5: Challenges of Scale-up of Cell Separation and Purification Techniques

5.1 Introduction
5.2 Scalable Cell Separation for Cell Therapy
5.3 Currently Developed Cell Separation Techniques
5.4 Conclusion
Acknowledgements
References

Chapter 6: Fundamental Points to Consider in the Cryopreservation and Shipment of Cells for Human Application

6.1 Introduction
6.2 The Role of Cryoprotective Agents (CPA)
6.3 Vitrification versus Cryopreservation
6.4 Points to Consider in the Development of Cryopreservation Protocols
6.5 Large-volume Freezing
6.6 Cryopreservation as Part of Manufacturing Processes
6.7 Conclusions
Acknowledgements
References

Chapter 7: Short-term Storage of Cells for Application in Cell-based Therapies

7.1 Introduction
7.2 Hypothermia and Mammalian Cell Storage
7.3 The Application of Hypothermic Storage in Cell-based Therapies
7.4 Concluding Remarks
References

Chapter 8: Cell Therapy in Practice

8.1 Introduction
8.2 The Classification of ATMPs
8.3 European Regulations
8.4 ATMP Case Study: Autologous Limbal Stem Cell Therapy: the Newcastle Experience
8.5 Conclusion
References

Index

End User License Agreement

List of Illustrations

Chapter 1: Overview of the Cell Therapy Field

Figure 1.1 Process flow diagram of the sequence of unit operations used in biopharmaceutical manufacture and their relative scales: (a) general process used for the manufacture of monoclonal antibodies; (b) manufacturing for a one on one therapy; and (c) manufacturing process for a one to many cell therapy process.
Figure 1.2 Heuristic classification of a selection of cell therapies to the three dimensions. Therapies shown in red are one to many, and therapies in blue are one to one. Numbers are our estimate of all stem cell trials in the clinical trials database that met our criteria. Therapies in boxes have received a market authorization. Abbreviations: EMA, European Medicines Agency; FDA, US Food and Drug Administration; HPC, human progenitor cells.
Figure 1.3 A comparison between 2011 and 2014 of the change in cell therapy clinical trials using www.clinicaltrials.gov and the search term “stem cells”. Hematopoietic stem cell clinical trials and those terminated before completion were excluded. The trials were analyzed using three dimensions, clinician-company, autologous-allogeneic and procedure-product.
Figure 1.4 Operations performed in upstream and downstream processing of cell therapies.
Figure 1.5 Cell banks are created for sustained manufacture of a cell therapy. From a master cell bank, working cell banks are developed which are then used in the manufacturing process.

Chapter 2: Structured Methodology for Process Development in Scalable Stirred Tank Bioreactors Platforms

Figure 2.1 Different impeller types used in stirred tank bioreactors (courtesy of EMD Millipore Corporation).
Figure 2.2 Different states of solid suspensions including on-bottom (a), just suspended (b) and uniform suspension (c) (courtesy of EMD Millipore Corporation).
Figure 2.3 Schematic summary of microcarrier screening for a specific stem cell line from initial screening in 2D low attachment Petri dishes to final microcarrier selection in spinner flasks and then proof of principle in Mobius^® CellReady 3L Bioreactor (courtesy of EMD Millipore Corporation).
Figure 2.4 Growth of BM-derived hMSCs in Mobius® CellReady 3L bioreactors at different seeding densities while keeping the impellor ON/OFF every 45 minutes for 2 cycles (courtesy of EMD Millipore Corporation).
Figure 2.5 Cell attachment efficiency at different seeding densities while keeping the impellor ON/OFF every 45 minutes for 2 cycles (courtesy of EMD Millipore Corporation).
Figure 2.6 Growth kinetics of hMSCs with different cells:bead ratios (courtesy of EMD Millipore Corporation).
Figure 2.7 Growth kinetics of hMSCs in both Mobius® CellReady 3L and 50L bioreactors (courtesy of EMD Millipore Corporation).
Figure 2.8 The glucose, glutamine, lactic acid and ammonia concentrations in culture over a 16-day culture period (courtesy of EMD Millipore Corporation).
Figure 2.9 Characterization of hMSCs produced from Mobius® CellReady 50L bioreactors (courtesy of EMD Millipore Corporation).

Chapter 4: The Scale-up of Human Mesenchymal Stem Cell Expansion and Recovery

Figure 4.1 (a) Cumulative cell numbers (upper) and population doublings (lower) at 100% dO₂ at saturation (20% O₂ v/v gas phase) (left) and 20% dO₂ at saturation (4% O₂ v/v gas phase) (right) with 100%, 50% and 0% medium exchange (mean value ± SD, n = 4); (b) The effect of medium exchange and oxygen concentration on metabolite consumption and production (mean value ± SD, n = 4) (Rafiq et al., 2013c).
Figure 4.2 Comparison of static and agitated conditions for hMSC microcarrier culture, and a comparison of different microcarriers in 100 mL spinner flasks. Glucose consumption and lactate production for hMSC1 (a), hMSC2 (b) and hMSC3 (c). Data is presented as mean ± SD (n = 4). Significant differences in values were noted with p < 0.05 (*), p < 0.01 (**) and p < 0.005 (***) in comparison to the highest respective value.
Figure 4.3 The two different impeller geometries that were used in the 100 ml (working volume) spinner flasks: the first, G1 (a) consisted only of a magnetically driven horizontally suspended flea (length 45 mm; diameter 10 mm), whilst the second, G2 (b), was made up from the same suspended flea with an additional vertical Teflon paddle (30 mm high; length 55 mm). Calculation of Kolmogorov microscale of turbulence, λ_K µm. Impeller G1 (Flask A) consisted only of a magnetically driven horizontally suspended flea (length 45 mm; diameter 10 mm), whilst impeller G2 (Flask B) had an additional vertical Teflon paddle (30 mm high; length 55 mm) (Hewitt et al., 2011).
Figure 4.4 The growth of hMSCs in 5 L bioreactor vessels over a period of 12 days. (a) Viable cells/ml readings for bioreactor 1 (left) and bioreactor 2 (right); (b) nutrient and metabolite concentrations for bioreactor 1 (left) and bioreactor 2 (right); (c) pH and dO₂ readings for bioreactor 1 (left) and pH and dO₂ readings for bioreactor 2 (right) (Rafiq et al., 2013a).
Figure 4.5 Phase-contrast image of microcarrier and cell suspension after dissociation during the novel harvesting procedure. Magnification 10x. Arrows: (a) Single hMSC successfully detached from microcarrier; and (b) hMSCs still attached to microcarrier (Nienow et al., 2014).

Chapter 5: Challenges of Scale-up of Cell Separation and Purification Techniques

Figure 5.1 Principle of fluorescence-activated cell sorting (FACS). After a single cell suspension is incubated with one or more fluorescently labelled antibodies, a sample is diluted in suspension buffer and run through the cytometer. Cells are hydrodynamically focused by sheath fluid added through a very small nozzle. The tiny “stream” of fluid takes the cells past laser beam(s) one cell at a time. A number of detectors detect the light scattered from the cells/particles as they go through the beam: one in front of the light beam (Forward Scatter or FS) and several to the side (Side Scatter or SS). Fluorescent detectors are used for the detection of fluorescence emitted from positively stained cells/particles (image provided courtesy of Abcam Inc, image copyright © 2016 Abcam).
Figure 5.2 Principle of magnetic-activated cell sorting (MACS). Cells in a single-cell suspension are magnetically labelled with microbeads and applied onto a column placed in a magnetic separator. Unlabelled cells pass through, while the magnetically labelled cells are retained within the column. The flow-through can be collected as the unlabelled cell fraction. After a short washing step, the column is removed from the separator, and the magnetically labelled cells are eluted from the column with a plunger. Thus, both labelled and unlabelled cell fractions can be isolated (image provided courtesy of Miltenyi Biotec, image copyright © 2014 Miltenyi).
Figure 5.3 Principle of acoustophoresis: (a) Photo of the acoustophoresis microfluidic system; (b) Cross-sectional view of cell distribution in the microchannel without ultrasound (left) and with ultrasound forming an ultrasound standing wave (right); (c) Illustration of acoustophoresis chip (reprinted from Burguillos et al., 2013).
Figure 5.4 Principle of immunoadsorption. pluriBead® is a cell separation technology that works without magnetic components: (1) pluriBeads (a) are added to a heterogeneous cell sample (b); and (2) bind target cells; (3) Next, the sample is sieved through a strainer, after which the pluriBeads with bound target cells stay on top, while unwanted cells pass through; (4) Target cells are then detached using a proprietary detachment buffer; and (5) collected (reproduced with permission of pluriSelect).
Figure 5.5 Principle of quadrupole magnetic cell sorting. Schematic drawing of the separation column of the Quadrupole magnetic cell sorter (QMS) showing cell injection port (a'), carrier (sheath fluid) injection port (b'), and elution of non-magnetically labelled cells (a) and magnetically labelled cells deflected by the magnetic field (b). The top view, B, shows the magnetic forces acting on the column to deflect cells. C is a photograph of the bench scale system. D is an enlarged diagram of the magnetic separation zone indicating the location of the inner and outer splitting cylinder (reproduced from Lara et al., 2006, Wiley Periodicals, Inc., copyright © 2006).
Figure 5.6 Principle of solution-based magnetophoretic separation. Schematic illustration of magnetophoretic cell separation using a magnetic repulsion force in a paramagnetic solution. When a magnetic field perpendicular to the direction of flow is applied by a permanent magnet placed next to the Ni microstructure, cells in a paramagnetic solution are deflected laterally due to the magnetic repulsion force. The separation efficiency is greatly enhanced by changing medium magnetic susceptibility from χm1 to χm2 via use of paramagnetic salt solutions (reprinted with permission from Shen et al. 2012, American Chemical Society, copyright © 2012).
Figure 5.7 Principle of optical tweezers. Optical tweezers use a strongly focused beam of light to trap objects. Intensity gradients in the converging beam draw small objects, such as a colloidal particle, towards the focus, whereas the radiation pressure of the beam tends to blow them down the optical axis. Under conditions where the gradient force dominates, a particle can be trapped, in 3D, near the focal point (Grier, 2003, reprinted by permission from Macmillan Publishers Ltd, copyright © 2003).
Figure 5.8 Principle of optoelectronic tweezers. Device structure used in optoelectronic tweezers. Liquid that contains microscopic particles is sandwiched between the top indium tin oxide (ITO) glass and the bottom photosensitive surface consisting of ITO-coated glass topped with multiple featureless layers: 50 nm of heavily doped hydrogenated amorphous silicon (a-Si:H), 1 mm of undoped a-Si:H and 20 nm of silicon nitride. The top and bottom surfaces are biased with an a.c. electric signal. The illumination source is a light-emitting diode operating at a wavelength of 625 nm (Lumileds, Luxeon Star/O). The optical images shown on the digital micromirror display (DMD) are focused onto the photosensitive surface and create the non-uniform electric field for DEP manipulation (Chiou et al., 2005, reprinted by permission from Macmillan Publishers Ltd, copyright © 2005).
Figure 5.9 Principle of optical chromatography. Schematic of particle motion under radiation pressure. The laser beam is introduced from the left-hand side and the liquid from the right-hand side: (a) particle introduction; (b) focus of particle into beam centre by gradient force; (c) acceleration of particle; (d) deceleration of particle; and (e) particles drifting at equilibrium position, resulting in particle separation. Particle elution is then achieved by decreasing the laser power (smallest particles elute first, followed by subsequent particles upon further decreasing the laser power (Imasaka et al., 1995, reprinted (adapted) with permission from American Chemical Society, copyright © 1995).
Figure 5.10 Principle of optical landscape fractionation. Low Reynolds number flows will be laminar: without an actuator all particles from chamber B would flow into chamber D. Chamber A would typically introduce a “blank” flow stream, although this could be any stream into which the selected particles are to be introduced. By introducing a 3D optical lattice – in this case a body-centred tetragonal (b.c.t.) lattice – into the fractionation chamber (FC), one species of particle is selectively pushed into the upper flow field. The reconfigurability of the optical lattice allows for dynamic updating of selection criteria. For weakly segregated species, the analyte can be either recirculated through the optical lattice or directed through cascaded separation chambers. This latter option also allows the use of multiple selection criteria in a single integrated chip (MacDonald et al., 2003, reprinted by permission from Macmillan Publishers Ltd, copyright © 2003).

Chapter 6: Fundamental Points to Consider in the Cryopreservation and Shipment of Cells for Human Application

Figure 6.1 Changes in cells during cryopreservation and vitrification processes.
Figure 6.2 Generic “bell curve” illustrating typical trend in cell survival with increasing cooling rates.

Chapter 7: Short-term Storage of Cells for Application in Cell-based Therapies

Figure 7.1 The potential for hypothermic storage in CTP logistics for autologous and allogeneic therapies.

Chapter 8: Cell Therapy in Practice

Figure 8.1 Purified human Islets of Langerhans as a cell therapy product.
Figure 8.2 Decision tree for the classification of cell therapy medicinal products.
Figure 8.3 ATMP manufacturing in a licensed facility (photo taken in the GMP production rooms of the Newcastle Cellular Therapies Facility).
Figure 8.4 Application of a sheet of autologous limbal stem cells that have been expanded ex vivo, from a limbal explant, on a sheet of human amniotic membrane as a substrate, following superficial keratectomy (debridement) of corneal pannus scar tissue.
Figure 8.5 Colour photographs of patient with unilateral total limbal stem cell deficiency (a) before autologous limbal stem cell transplantation, and (b) 12 months after transplantation.
Figure 8.6 Implantation of the first limbal stem cell product in our Phase II clinical trial.

List of Tables

Chapter 2: Structured Methodology for Process Development in Scalable Stirred Tank Bioreactors Platforms

Table 2.1 k_La studies with microcarriers done in Mobius® CellReady 3L bioreactors.
Table 2.2 Microcarrier property information.
Table 2.3 Cost comparison of 10 million cells in Mobius® CellReady 3L and 10-stack CellStack® Plate.
Table 2.4 Cost breakdown analysis.

Chapter 3: The Effect of Scale-up on Cell Phenotype: Comparability Testing to Optimize Bioreactor Usage and Manufacturing Strategies

Table 3.1 Cell comparability in the development path.
Table 3.2 Example of required cell numbers for several cell therapy applications.
Table 3.3 Overview of possible cell characterization assays.

Chapter 4: The Scale-up of Human Mesenchymal Stem Cell Expansion and Recovery

Table 4.1 A risk/reward and cost comparison of the scale-out (automation) or scale-up (bioreactor) options.
Table 4.2 Physical aspects of the agitation/agitator requiring consideration and biological aspects that are often system specific (Hewitt and Nienow, 2007).
Table 4.3 (a) Impact of oxygen concentration and medium exchange on specific growth rate, fold increase and doubling time. 100% dO₂ values are emboldened and underlined, whereas 20% dO₂ values are not; (b) Impact of dissolved oxygen concentration and medium exchange on specific glucose consumption, lactate production and yield of lactate on glucose. Negative values indicate consumption. 100% dO₂ values are emboldened and underlined, whereas 20% dO₂ values are not (Rafiq et al. 2013c).
Table 4.4 Properties of commercially available microcarriers.
Table 4.5 Summary of the mean maximum number of cells achieved per Cytodex 3 microcarrier under various conditions using both types of 100 ml spinner flasks G1 (Flask A from Figure 4.3) and G2 (i.e. Flask B from Figure 4.3) (Hewitt et al., 2011).

Chapter 5: Challenges of Scale-up of Cell Separation and Purification Techniques

Table 5.1 The various cell separation methodologies set against 4 parameters, and scored for cell manufacturing potential based on type of production (batch or continuous) and system (closed or open)

Chapter 7: Short-term Storage of Cells for Application in Cell-based Therapies

Table 7.1 Parameters and cell viability outcomes for the short-term storage of MSCs

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List of Contributors

Che J. Connon
Institute of Genetic Medicine
Newcastle University
Newcastle upon Tyne
UK

Karen Coopman
Centre for Biological Engineering
Loughborough University
Leicestershire
UK

Julian R. De Havilland
Cellular Therapies Facility
Newcastle University
Newcastle upon Tyne
UK

Paul A. De Sousa
Centre for Clinical Brain Sciences and
MRC Centre for Regenerative Medicine
University of Edinburgh
Edinburgh
UK

Gustavo S. Figueiredo
Department of Ophthalmology
Royal Victoria Infirmary
Newcastle upon Tyne
UK

Institute of Genetic Medicine
Newcastle University
Newcastle upon Tyne
UK

Francisco C. Figueiredo
Department of Ophthalmology
Royal Victoria Infirmary
Newcastle upon Tyne
UK

Institute of Genetic Medicine
Newcastle University
Newcastle upon Tyne
UK

Lucy Foley
Centre for Process Innovation – Biologics
Darlington
UK

Jason Hamilton
Athersys Inc
Cleveland, OH
USA

Lyn Healy
Haematopoietic Stem Cell Laboratory
The Francis Crick Institute
London
UK

Thomas R. J. Heathman
Centre for Biological Engineering
Loughborough University
Leicestershire
UK

Christopher J. Hewitt
Centre for Biological Engineering
Loughborough University
Leicestershire
UK

Aston Medical Research Institue
School of Life and Health Sciences
Aston University
Birmingham
UK

Marieke A. Hoeve
Centre for Clinical Brain Sciences and
MRC Centre for Regenerative Medicine
University of Edinburgh
Edinburgh
UK

Charles J. Hunt
UK Stem Cell Bank
Advanced Therapies Division
National Institute for Biological Standards and Control
Hertfordshire
UK

Donghui Jing
HaoLing Cell Technologies Corporation
TEDA
Tianjin
P.R.China

Daniel Kehoe
Stem Cell Bioprocessing Group
Process Solutions
EMD Millipore Corporation
Bedford
USA

Majlinda Lako
Institute of Genetic Medicine
Newcastle University
Newcastle upon Tyne
UK

Jennifer Man
UK Stem Cell Bank
Advanced Therapies Division
National Institute for Biological Standards and Control
Hertfordshire
UK

John Morris
Asymptote Ltd
St John's Innovation Centre
Cambridge
UK

Julie Murrell
Stem Cell Bioprocessing Group
Process Solutions
EMD Millipore Corporation
Bedford,
USA

Alvin W. Nienow
Centre for Biological Engineering
Loughborough University
Leicestershire
UK

Centre for Bioprocess Engineering
University of Birmingham
UK

Qasim A. Rafiq
Centre for Biological Engineering
Loughborough University
Leicestershire
UK

Aston Medical Research Institue
School of Life and Health Sciences
Aston University
Birmingham
UK

Glyn N. Stacey
UK Stem Cell Bank
Advanced Therapies Division
National Institute for Biological Standards and Control
Hertsfordshire
UK

Stephen Swioklo
Institute of Genetic Medicine
Newcastle University
Newcastle upon Tyne
UK

Bart Vaes
ReGenesys BVBA
Heverlee
Belgium

Huaqing Wang
Department of Medical Oncology
Nankai University Cancer Hospital
Tianjin Union Medical Center
Tianjin
P.R.China

Stephen Ward
Cell and Gene Therapy Catapult
Guy's Hospital
London
UK

Michael Whitaker
Institute of Cell and Molecular Sciences
Newcastle University
Newcastle upon Tyne
UK

Nicholas A. Willoughby
Institute of Biological Chemistry
Biophysics and Bioengineering
School of Engineering and Physical Sciences
Heriot-Watt University
Edinburgh
UK

Preface

Cell-based therapy is an exciting and rapidly developing field of medicine for the 21st century. However, this new paradigm brings new challenges in regulation and production, which requires cross-disciplinary approaches and greater collaboration between clinicians, academics and industrial scientists. This new alliance is reflected in the range of disciplines from which the distinguished contributing authors have been drawn to create this book, including Industry (EMD Millipore Corp., Athersys Inc., ReGenesys, Asymptote Ltd), Governmental (Process Innovation Centre – Biologics, Cell Therapy Catapult, UK Stem Cell Bank) and Academic Institutions (The Francis Crick Institute, and Newcastle, Birmingham, Loughborough, Aston, Edinburgh and Heriot-Watt universities). Together they represent the cutting edge, industrially focused frontline of bioprocessing for cell-based therapies.

The book is composed of eight chapters beginning with an introductory chapter that sets out the history of cell therapy leading up to the current and future challenges associated with the manufacture and distribution of this relatively new class of therapeutic modality. This includes a detailed discussion on the advantages and disadvantages of the one-one and one-many approaches in cell-based therapy. The book then moves into a series of chapters focusing on recent technical developments in bioprocessing for cell-based therapies. Chapter 2 discusses the use of stirred tank bioreactors for hMSC cultivation, specifically engineering requirements such as mixing phenomena, oxygen and heat transfer rates, metabolic demands of the cells and particular problems associated with adherent cells. Chapter 3 follows this theme by focusing on the important topic of cell characterization during scale-up of hMSCs. We are informed that to guarantee product consistency there needs to be uniformity in identity, maintenance of a unique product character and crucially demonstration of a consistent and therapeutically relevant product potency. Chapter 4 also focuses on hMSC culture, asking if we need to consider scale-up or scale-out. This chapter, then proceeds to highlight the technical difficulties involved in moving bioprocessing research from the lab-scale to large-scale, covering the challenges associated with choice of microcarriers and spinner flasks, then removing cells from chosen microcarriers. Chapter 5 continues by describing the importance of cell separation for cell-based therapies with an in-depth focus on the current methods for mammalian cell separation and their suitability for application in large-scale cell purification for clinical application. We then move on to Chapters 6 and 7, which discuss the importance of storage and transport of the cellular products. Chapter 6 focuses on cryopreservation, how to limit cell damage, large volume freezing, approaches to biobanking and regulatory requirements. Chapter 7 takes an interesting departure from cryopreservation, instead focusing on cold or hypothermic cell preservation, in which the logistics of short-term storage are described as are the use of hydrogels as a novel storage medium. The final chapter (Chapter 8) focuses on where the advancements in bioprocessing end up, such as in the clinical application of cell-based therapies. This chapter includes a case study describing the development of a new therapy against the background of a rapidly changing regulatory environment and the challenges this poses, before bringing the therapy successfully to the clinic.