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STRATEGIES FOR SCALE-UP & SCALE-OUT

EXPERT INSIGHT Scale up of production from human pluripotent stem cells for developing targeted therapies: advances and challenges

Jonathan N Thon & Sven M

Without question, the future of regenerative medicine is in the scalable production of transfusable human tissues for therapeutic use. will be among the first of these stem-cell based therapeutic tissues to be developed and adopted for clinical use, most notably because they are anucleate and can be safely irradiated to substantially reduce the risk of teratoma development and other cell contaminants. Furthermore, plate- lets are short-lived, well characterized, easily transplanted, are not re- quired to be autologous, and support a larger than $20 billion per year global market that relies entirely on human volunteer donors. While we are within a decade of realizing the potential of stem cell-based therapeu- tics, this was not obvious only several years ago. This article identifies the major logistical challenges associated with commercially scaling platelet production for therapeutic use and our experiential insights into translat- ing this technology to the clinic. Submitted for review: Sep 6 2017 u Published: Nov XX 2017

While platelets are primarily re- immunity [1–6]. Among the stem be safely irradiated to kill any con- sponsible for clot formation at sites cell-based therapeutic landscape, taminating nucleated cells, thereby of active hemorrhage, it is becom- platelets are an ideal early entrant reducing the risk of teratoma devel- ing increasingly apparent that they because they: are in high clinical de- opment [8,9]; do not require HLA or also play significant roles in wound mand due to their short inventory matching for the major- healing, angiogenesis and innate shelf-life [7]; are anucleate and can ity (>90%) of platelet transfusions,

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which will facilitate large-scale for , tissue regenera- manufacture of allogeneic human tion, wound healing, drug delivery, pluripotent stem cells (hPSCs) for and there is even work in the cos- off-the-shelf therapy (Box 1); are metics industry using platelets for short-lived and well characterized, skin rejuvenation [16–28]. Platelet which will simplify projected clin- BioGenesis is addressing this need ical trials [10]; are easily transplant- by establishing a scalable, current ed [11]; and would benefit tremen- good manufacturing practice (cG- dously from sterile manufacture, as MP)-compliant commercial plat- current donor-based platelet trans- form to make safe and functional fusions are inherently susceptible to human platelets [29]. In this arti- bacterial and viral contamination cle, we describe the challenges in [12,13]. bringing hPSC-platelets from the Current platelet demand exceeds research phase through to com- supply by ~20% [14], and unmet mercially viable products. We lay demand is expected to more than out best practices for overcoming double by 2022 due to a growing these obstacles, ensuring regulatory and aging population requiring compliance, establishing a scalable more platelet-based procedures, process, and securing freedom to new medications that reduce plate- operate. We conclude with trans- let counts, and the expansion of lational insights into the future of existing uses of platelet transfusions regenerative medicine and the mar- to improve healing time [15]. In the ket expansion of stem cell-derived USA, the blood market effectively platelet products. operates as an oligopoly, with the While all platelets are hPSC-de- American Red Cross and Ameri- rived, for the purposes of this article ca’s Blood Centers each controlling hPSC-derived platelets will refer to a little less than half of the market platelets generated outside the hu- and HemeXcel as the third major man body by industrial cell culture. blood provider. There are numerous other potential markets for plate- lets and their growth factors that are currently constrained by lack COMMERCIALIZATION of access to platelets. For example, CHALLENGES platelet growth factors can be used There is a tremendous amount of ffBOX 1 variability among donated plate- lets and the various quality assess- Platelets synthesize class I HLA (but not class II). While platelets ab- ment metrics that are used to gauge sorb soluble HLA antigens from plasma, leading to relatively high- platelet function [30]. Despite this er numbers of class I HLA antigens on their surface as compared to red cells and , the incidence of HLA antibodies follow- fact, there has been a historical pre- ing is low (10–20%) [71,72]. Interestingly, >50% sumption that donor-derived plate- of patients with HLA antibodies fail to manifest refractoriness, such lets ‘work’, and there is no single that the incidence of HLA-mediated refractoriness following platelet gold-standard assay to assess donor transfusion is only 3–5% [71]. platelet unit quality and function Serum-free production of platelets should result in lower num- ex vivo ahead of transfusion. Instead, cur- bers of class I HLA antigens on their surface potentially resulting in a lower incidence of HLA-refractoriness still. While this prediction will rent US Food and Drug Admin- need to be borne out in the data, it argues strongly against the need to istration (FDA) requirements re- invest in the production of HLA-null (universal) platelets as a first-tier garding platelet unit preparation product. focus on storage temperature, pH,

702 DOI: 10.18609/cgti.2017.068 expert insight residual leukocyte count and bac- ff Establishing a consistent terial/viral contamination rate. To differentiation protocol that avoids serum or feeder-cells, license platelets based on post-stor- which would likely make age radiolabeled autologous platelet regulatory approval substantially viability measurements in normal more challenging and add variability and contamination risk subjects, the FDA requires that the to the product. lower post-storage 95% confidence ff Developing an industrial limits (LCLs) for platelet recoveries process during early stages of are ≥66% and survivals are ≥58% of development that will scale to the same subject’s radiolabeled fresh meet future clinical and then commercial demand for hPSC- recoveries and survivals, respectively derived platelets, so that the [31]. While donors volunteer plate- process does not need to be lets, procurement of platelet units is redeveloped at each stage. not free. Blood bankers, who are the ff Prioritizing first quality and key decision makers for platelet or- then cost, so that hPSC-derived platelets will meet regulatory ders and distribution, choose their requirements and result in a platelet supplier based on availabil- product that is commercially ity and price (Box 2). Therefore, in viable. addition to demonstrating non-in- The tradeoff between focusing feriority relative to donor platelets, on quality and cost is the principal hPSC-derived platelets need to be challenge for regenerative medicine cost-competitive to warrant com- and a significant barrier to creating mercial adoption. a scalable and profitable business The improved safety, projected in this space. While regenerative ‘on-demand’ availability, function- medicine is expected to ultimately al definition of product, and low- improve patient safety and reduce er inter-unit variability constitute overall costs, PSC-derived prod- the major value propositions that ucts are likely to be more expensive will drive market adoption of hP- in the short term. It will therefore SC-derived platelets over donor require all stakeholders working platelets. Additionally, the reduced together to support a commercial inter-unit variability will substan- path to market for early regenera- tially empower physicians to begin tive medicine technologies such as predicting how donors will respond platelets, by supporting transforma- to platelet transfusions. Commer- tive advancements in this space and cial development of hPSC-derived providing reimbursement rates that platelets should therefore strive to reflect these long-term benefits. leverage the value propositions of donor independence (sterile, safe, and scalable products) and on-de- mand manufacturing capability by: REGULATORY ff Advancing a donor-independent, COMPLIANCE cGMP-compliant hPSC line Current Good Manufacturing Prac- rather than developing processes around research grade or animal tice comprises a set of standards lines that cannot be advanced that are set forth by the FDA that clinically, or relying on primary describe ways that manufacturing CD34+ stem cells that carry the same availability, sterility and processes and facilities must be cost limitations as donor-derived designed, overseen, and operated. platelets. Observation of cGMP during the

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ffBOX 2 ff Clinical development plan, A leukoreduced, irradiated platelet unit in the United States including: costs approximately $1000 when accounting for the cost of discard- ffPhase 1 clinical study synopsis

ed units. Leukoreduced (non-irradiated) platelet units are marginally ffPhase 2 clinical study synopsis cheaper, averaging just under $750 per unit and comprising an addi- tional ~50% of all platelets transfused. Prices vary greatly depending While this is an ever-changing on supply and demand [73–76]. landscape, there are guiding prin- ciples that will need to be consid- ered for advancing hPSC-derived manufacture of hPSC-derived ther- platelets. Most differentiation pro- apeutics ensures that the consum- cesses are developed in primary er receives products of the correct hPSC-lines or animal lines because identity, strength, quality and puri- of accessibility and cost, but it is ty. In order to be cGMP-compliant, critical to note that cost or reg- the responsibilities of the manufac- ulatory compliance may prohib- turer of an hPSC-therapeutic in- it their commercial advancement clude obtaining appropriate quality and pre-clinical validation studies raw materials, establishing robust will need to be re-established us- operating procedures, establishing ing a cGMP-compliant line. This strong quality management systems, is especially true of research-grade detecting and investigating product PSC-lines, as different PSC-lines quality deviations, and maintaining will perform very differently using reliable testing laboratories. the same differentiation protocol For hPSC-derived platelet pro- [36,37], which will add substantial duction, the product will need to time, cost and risk to the research meet a number of regulatory re- and development strategy. quirements in order to gain inves- The potential immunological tigational new drug (IND) approval response to a stem cell generated [35]. This includes compiling a de- therapeutic, which could be driv- scription of the: en by either the hPSC-line itself or ff Proposed indication products used during the differen-

ff Dosage and route of adminis- tiation process, is equally import- tration ant to consider. Human leukocyte

ff Product description and chem- antigen (HLA) is a polymorphic, istry manufacturing controls cell surface protein that is expressed (CMC), including: on the majority of nucleated cells ff iPSC line derivation and mediates immunoreactivity to

ff Master cell bank production transplanted tissues [38]. If the im-

ff production mune system identifies cells express- ing HLA molecules that it deems to ff Platelet production be ‘foreign’ within the body, it will ff Product characterization, mount an immune response that including: can lead to graft rejection or com- f f iPSC cell line characterization plications following transfusions or ff Megakaryocyte characterization­ transplants [38]. Therefore, HLA ffPlatelet final product compatibility must be taken into char­acterization account during the selection of an ff Storage characterization hPSC-line. This is less of a prob- ff Description of pre-clinical studies lem with platelets, but this can be

704 DOI: 10.18609/cgti.2017.068 expert insight a major issue with other cells/tissues being incorporated into the differen- (Box 1). One risk-mitigating ap- tiation protocol are available in cG- proach is to begin with hPSC-lines MP-compliant versions. The use of derived from HLA SuperDonors, different versions of reagents (both which have HLA genetic profiles within and between manufacturers) that make their cells less likely to can cause variability in cell culture cause immune complications in outcomes, resulting in the need for platelet recipients [39]. The basis additional optimization steps. This for this is a partial HLA match with can add unnecessary time and cost most recipients that has been shown to the process of translating a stem to be beneficial in organ transplants cell culture process to the clinic. [40]. Indeed, hPSCs have been Similarly, it is important to estab- successfully generated from HLA lish supplier relationships in order SuperDonors using a proprietary to ensure that the required products transgene-free and virus-free episo- can be made available at clinical or mal reprogramming process that commercial quantities and at a cost utilizes circular DNA vectors to that supports the scalability of the deliver pluripotency genes driving process. human induced PSC production In order to ensure that time is [41–46]. As this reprogramming not wasted unnecessarily, it is also method does not rely on integra- important to establish a scalable tion events, it alleviates major safe- manufacturing process that can be ty concerns about potential use of performed in a cGMP-compliant hPSC-derived products in cellular manner. If this is not done initial- therapies (e.g., genetic integration ly, the process will need to be re- of the transgene vector into off-tar- developed and approved prior to get genes with negative consequenc- clinical trials and again at each scale es, escape of the virus carrier, etc.) up stage, adding substantial time [34,47], and it is recommended for and cost to the development and making platelets. approval process. This point was Once the appropriate stem cell recently made and elaborated upon line is identified, the differentiation in a FDA guidance document [48]. process also needs to be adapted to This also requires the establishment be cGMP-compliant. Stem cell cul- of supplier relationships that afford tures typically rely on embryonic access to cGMP-compliant biore- fibroblast feeder cells that can po- actors at commercial and clinical tentially be contaminated with xe- volumes that support scalability of nogeneic pathogens, increasing the process in a cost-effective manner. risk for an immunogenic reaction in humans. Alternatives to animal serum and feeder cells are desirable to avoid the introduction of animal SCALABILITY OF PROCESS products into human tissue culture, The regenerative medicine market’s to reduce the harvesting of cells or rapid growth is projected to con- serum from animal fetuses, and to tinue and is estimated to exceed ensure safe and animal product free $38 billion within 5 years. Stem conditions for cGMP [28]. cell-based therapeutics will require Additionally, it is important to the continuous generation of tril- ensure that any reagents that are lions of hPSCs and their resultant

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differentiated cells. However, cur- be expanded in vitro to generate an rent laboratory-scale tissue cul- adequate number or precursor cells. ture methods using culture dishes Secondly, hiPSCs must be differen- or spinner flasks cannot produce tiated into functional megakaryo- clinical-grade cells at this scale in cytes that are themselves capable of a robust and cost-effective manner. platelet production. Thirdly, mega- The outcome is an inability to scale karyocytes must be triggered to pro- processes linearly, which, along with duce platelets. Finally, the platelets the cost of cell culture media, con- must be isolated and concentrated stitutes a major limiting factor to into transfusable units (Figure 1). market growth. Laboratory scale Many of the challenges in pro- production needs to be replaced ducing stem cell-derived products by industrial-scale processes, which are illustrated by the production of means that technology relying on platelet generating megakaryocyte small-scale bioreactors must be re- cells, which constitutes the first two developed in larger volume systems phases of industrial platelet produc- that maximize efficiency and yield tion. Like many hPSC-derived cell while minimizing cost. types, megakaryocyte generation Platelets are produced by large, from hPSCs comprises multiple polyploid, PSC-derived cells called differentiation steps, which include that reside adjacent both adherent and soluble phases to sinusoidal blood vessels in the and different media requirements. bone marrow. During thrombopoi- Additionally, the cells are sensitive esis, megakaryocytes generate bead- to shear stress, there are major size like extensions called proplatelets differences between the immature that enter these vessels and sequen- and differentiated cells, and there is tially release platelets into the circu- a need to isolate specific cell inter- lation. Whereas bone marrow mega- mediates at multiple stages within karyocytes generate >2,000 platelets the process. This requires the serial each within <12 hours, in vitro hP- linking of multiple differentiation SC-derived megakaryocytes in static stage-specific bioreactors together culture generate <10 platelets each to enable scaled up industrial pro- over 48–72 hours [49]. To improve duction. While complex produc- the efficiency of stem cell based tis- tion processes such as this have been sue production, we should follow a successfully implemented in a few key guiding principle for regenera- cases (e.g., cardiomyocytes [50,51]), tive medicine and look to the body production limitations have pre- for inspiration on how to reproduce vented the clinical advancement physiologic processes in an industri- of many other hPSC-derived cell al setting. The overarching goal in types (including megakaryocytes), this is to identify minimum viable and remain a major hurdle in the triggers for directed differentiation. commercialization of hPSC-derived In the case of platelet production platelets. Given the complexity of and achieving increased total plate- the process and its parallels to the let yield, anatomical platelet pro- generation of other stem cell derived duction can be translated into four tissues, solutions developed here are major phases of industrial cell cul- expected to be broadly applicable to ture that mimic what occurs in the general cell culture for tissues that bone marrow. First, hiPSCs must others are working to produce.

706 DOI: 10.18609/cgti.2017.068 expert insight

ffFIGURE 1 Phases of platelet production and challenges with industrial scale production at each stage.

pansin ierentiatin Trigger platelet Islatin prtin nentratin

Cerialiatin hallenges

eglatry pliane

Salaility press

ree t perate

Industrial-scale production of platelets requires the optimization of four phases cell-culture processes that mimic anatomical platelet production: expansion of hiPSCs to generate adequate numbers of progenitor cells, differentiation of precursor cells into functional megakaryocytes, triggered production of platelets by megakaryocytes, and isolation and concentration of hiPSC-derived platelets. In order to successfully commercialize hiPSC-derived platelet products, freedom to operate and maintenance of regulatory compliance are necessary at each stage of development. Each stage has unique challenges when it comes to achieving scalability of the process and overcoming commercialization challenges.

While much progress has been 6x1017 platelets [49], which rep- made in the development and op- resents a major manufacturing timization of these processes to challenge. Ways in which we can generate functional platelets from begin addressing these needs and hPSCs, up to now this has been optimizing phases three and four performed exclusively at a small of industrial platelet production scale. The next major challenge are two-fold: first, we must achieve in the translational application of greater yields of product cells from hPSC-derived platelets will be to progenitors (e.g., we must be able transition from laboratory scale to to isolate more megakaryocytes per industrial scale platelet production. hPSC and more platelets per mega- One platelet apheresis transfusion karyocyte); and secondly, we must unit contains ~3x1011 platelets in develop and optimize the physical 300–500 mL [49]. In the USA, infrastructure to support the gen- approximately 2 million aphere- eration of these numbers of cells sis-equivalent platelet units are re- and the media necessary to pro- quired annually, or approximately duce them.

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To date, most of the platelet bio- exposure of all cells, regardless of reactors that have been developed their position in the device. This have been custom-made, proof uniformity would allow for condi- of concept research tools that are tions to be precisely modulated in low-throughput and only capable order to identify ‘sweet spot’ rang- of producing platelets at a labora- es for individual drivers of platelet tory scale. During the transition to production. Computational fluid the fourth phase of industrial scale dynamics modeling within the bio- platelet production, several new reactor will be necessary to resolve approaches will be required during flow lines during device operation bioreactor development and man- and identify actual forces at play ufacture to enable the scalability on cells at different positions on the of the production process. Firstly, device, which could then be modu- platelet bioreactors capable of com- lated to meet these goals. mercial scale production must be manufactured using standardized Use of biocompatible processes and biocompatible mate- (non-PDMS) scalable rials that allow for automation. Sec- materials ondly, it is critical that we establish PDMS is commonly used for mi- standardized operational processes crofluidic device prototyping be- and device validation metrics that cause many of its properties make will meet regulatory requirements it ideal for device development and be cost effective, and that the and affordable manufacture [53]. failure rate for manufacture be very However, it is not suitable for use low [52]. in commercial biomedical devices Within these operational con- and it is critical that we transition straints, the following major ad- to biocompatible FDA-approved vancements that still need to be thermoplastics during the devel- achieved in the development of opment of commercially scalable next-generation millifluidic devices platelet bioreactors. PDMS can including: absorb small molecules and in- troduce uncross-linked oligomers Automation of flow control from the curing process into solu- Commercial scale platelet bioreac- tion [54,55], which can result in tors must be able to be run without cytotoxicity and can affect critical direct human supervision. These cell signaling dynamics and drug devices must be capable of running concentration studies. PDMS is in parallel over continuous process also unsuitable for high-through- conditions that account for the put manufacturing methods such pressure differentials that are gener- as injection molding, rolling and ated in devices large enough to meet embossing [56], preventing scalable projected scale requirements. manufacture. Alternative scalable materials that could be used in Bioreactor computational next-generation platelet bioreac- fluid dynamics modeling tors could include acrylic, cyclic An optimally designed bioreactor olefin copolymer, polycarbonate, should distribute cells uniformly polyvinyl chloride, polyolefin, and ensure equal agonist (e.g., shear polyethylene, polyurethane, poly- stress, extracellular matrix protein) propylene or graphene oxide.

708 DOI: 10.18609/cgti.2017.068 expert insight

Scaled design that supports devices. However, it is also necessary commercial application to allow for flexibility in production In order to perform preclinical stud- to adapt to expected future scientif- ies, including those needed to assess ic advances and market changes. platelet safety and quality in vitro Media volume reduction & and to perform functional studies in product concentration mice (Tables 1 & 2), platelet bioreac- tors must be scaled up to support the At present, a major bottleneck in production of at least 3x106 mega- all phases of industrial-scale plate- karyocytes to produce the required let manufacture is cost, which is 3x108 platelets [35,49,57]. The bio- largely driven by cell culture media. reactors will need to be scaled up Culture phases typically extend for further prior to commercialization multiple days and require the opti- to accommodate at least 3x109 mization of growth factors and dai- megakaryocytes to produce 3x1011 ly media exchanges. ‘Batch-feeding’ platelets, the equivalent of one media change approaches involve apheresis platelet unit, and the mil- siphoning off the majority of the lions of platelet units that are need- media and replacing it with fresh ed for use globally. Furthermore, media daily. This approach is not access to platelets today is currently ideal for stir tanks and other large restricted to transfusion patients in 3D bioreactors because it requires major cities in developed countries. that the stirrer be stopped for a peri- There is significant unmet demand od of time to allow the cells to settle by patients in the developing world to the bottom of the tank prior to and from other industries, such as removal of the old media, making cosmetics and orthopedics, which it cumbersome and expensive. Ad- desire access to the regenerative ef- ditionally, the shock of the media fects of their high concentration of exchange has unknown biological growth factors. These industries and impact on the cells. An alternative their platelet demand are expected to batch feeding is utilizing a mil- to grow further upon the availabili- lifluidic perfusion bioreactor which ty of hPSC-derived platelets and the employs real-time sensors to contin- development of novel applications uously replace media with appropri- of platelet products. ate supplements to replenish small molecules that are depleted over Scaled manufacture & time in culture [58–60]. Cells are re- assembly of devices tained in the bioreactor during the Current production techniques media exchange by a porous filter for platelet bioreactors should be or other cell retention device. Criti- modified to introduce scalable de- , perfusion bioreactors can per- sign principles that will reduce in- form these media exchanges while ter-batch variability during testing maintaining low, controllable and and will eliminate the need for uniform shear and pressure on cells. device redesign and revalidation in Incorporating media recirculation the future. The use of microinjec- loops in millifluidic device design tion molding, rolling or embossing will help to minimize operation- techniques will allow for the even- al volume, reduce media/cytokine tual large-scale reproducible manu- cost, and further concentrate the facture and automated assembly of cell product. Platelets must remain

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ff TABLE 1 Targeted platelet quality assessment metrics. General attribute Specific attribute Targeted perfor- ǂActivated (0.1 U/mL In vitro assay mance specification TRAP/1μg/mL CRP/20 Resting μM ADP)

Morphology Size 1.5–3.0 μm – Light Microscopy Shape Circular or flat/spread Discoid Cytoskeletal β1 tubulin Cortical coil of Centralization organization F-actin 6–8 MTs Central Fluorescence network Lamellipodia & filopodia microscopy formation Granule content PF4 (alpha granule) 40–80 Fuse with plasma Fluorescence Serotonin (dense 2–7 membrane microscopy granule) Release content Ultrastructure Membrane conformation Smooth Ruffled Electron microscopy Glycogen granules – Organelle content Present –

Open canalicular system 2–8 mitochondria – Dense tubular system Present –

Present

Biomarker Expression CD41/61 >80% >80% Flow cytometry PAC-1 <20% ≥80% CD42b >80% >80% CD62P <20% ≥20% CD63 <20% ≥20% Annexin V (5 mM CaCl2) <10% ≥10% Aggregation Aggregation – ≥120% slope Aggregometer ≥95% Aggregation amplitude – (+10 μg/mL Collagen) Clot retraction Clot retraction - Grade 3 or higher at 2 Glass test tube, hours plasma (measured at 2 and 24 Grade 4 or higher at 24 hours) hours In vitro assay ǂActivated (colla- ǂActivated (collagen, Fg, gen, Fg, vWF) vWF) Thrombus formation Extent of thrombus + +PLT-depleted blood Perfusion chamber formation – low vs high 30 minutes 30 minutes shear (calciein-AM) In vivo assay Circulation time/ Circulation time 8 hours – clearance ≥66/≥58% – Infusion into human- Platelet recovery/ ized vWF mice survival

710 DOI: 10.18609/cgti.2017.068 expert insight

ff TABLE 2 Targeted platelet safety specifications. Platelet metabolic Units Acceptable Target Allowable process failures activity metrics range

Storage temperature °C 22–24 With continuous gentle agitation during storage Actual platelet yield of x1011 ≥3.0 95/75%* N = 11** N = 18** N = 23** transfusable component 0 1 2 pH at 220C x1011 ≥3.0 95/75%* N = 60** N = 93** N = 124** 0 1 2 0 1 2 Residual leukocyte pH ≥6.2 95/95%*** count (95% <5x106 of units) Bacterial contamination ≤1:5000 95% rate *95% confidence that greater than 75% of the components meet the standard. **The sample size numbers can be used in a samploing plan that should be representative of products collected on each machine type in each facility. ***95% confidence that greater than 95% of the components meet the standard. agitated and in suspension to main- the product, but also the process, tain their functionality, which can which means that pre-IND valida- be achieved through the introduc- tion studies need to be performed tion of peristaltic pumps [61,62], using a cGMP compliant process which have the additional advan- that will remain consistent during tage of being able to recapitulate the clinical-grade product manufactur- physiologic pulsatile flow of ~1 Hz ing. While process changes can be [63,64]. We have had success imple- made, this requires reassessment by menting this approach in hPSC-de- the regulatory agencies and approv- rived platelet production processes, al of the change by the FDA prior with the potential to expand it to to implementation into clinical other large-scale, general cell culture manufacture. efforts[35,49,65] . In parallel to the advances that are being made on technological and process optimization, it is crit- FREEDOM TO OPERATE ical that a regulatory strategy is de- In order to successfully commer- veloped in collaboration with NHL- cialize a regenerative medicine tech- BI, NIAID, DoD, BARDA and the nology, it is not only necessary to FDA. These strategies should es- overcome the scientific challenges tablish industry standards for qual- presented with the technological de- ity and functional assessment of velopment but to also obtain com- cell-based therapeutics, operational mercial freedom to operate. Ideally, process standards, and bioreactor this includes control over the entire monitoring methods that will fit supply chain, and for hPSC-de- into larger manufacturing process- rived platelet production this can es. The FDA does not just approve be consolidated into three critical

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families of intellectual property (IP) space. Ownership of foundational [35]. The first is development of or patents gives a significant advantage license to a clinical grade hPSC line to first movers, effectively blocking for commercial therapeutic use. any other entrant from commercial- The second is a cGMP compliant izing a competing product. Moving process to generate platelet produc- early to obtain freedom to operate ing-megakaryocytes that is matched (or even better, to control the IP to the clinical grade hPSC line. landscape) will ensure a company Third, one must develop a scalable, has an indisputable competitive cGMP-compliant method of trig- advantage and creates a path for a gering megakaryocytes to produce project to be profitably financed by platelets (such as a platelet bioreac- private investors. Furthermore, as tor). As the field progresses it should new discoveries are made that build be presumed that at each step with- on the foundational IP, the oppor- in the differentiation process there tunity set will grow, and these early will exist the opportunity to include companies and their investors will additional technologies that can reap the rewards of the risks taken. further improve the cost and quali- ty of cells produced. However, these opportunities should be considered carefully in light of the associated li- TRANSLATIONAL INSIGHT censing expenses that can offset the The regenerative medicine industry benefits and can limit the commer- is still in its infancy, with rapidly cial viability of the product. Tech- evolving technology and near lim- nology licenses typically contain a itless possibilities for its application. number of financial terms, includ- This represents a huge opportunity ing an up-front fee, milestones pay- that companies establishing them- ments, annual license payments and selves today will be best positioned royalties. For sufficiently large tar- to capitalize upon. In financial get markets (such as platelets), the terms, investors should think of cost of one-time cash payments can backing these technologies as buy- be amortized over a very large num- ing an option: there is significant ber of units to become negligible. value to the optionality inherent in Royalties present a more substantial a young industry – if you invest in issue in regenerative medicine prod- teams capable of executing on this ucts that are high volume but rel- optionality. Investors and compa- atively low margin (like platelets). nies who are late to the game risk Even minor royalties can stack up missing the opportunity to capture and can quickly consume the prof- these significant markets(Figure 2). it margin. This will stall product This option value is already being development and destroy signifi- realized in the platelet space, with cant value that could be shared by applications extending beyond the all partners and, most importantly, initial focus on tissue regeneration prevent life-saving technology from into matters of national securi- reaching patients. ty, drug delivery and personalized In emerging industries such as medicine. regenerative medicine, the initial IP National defense and security filed tends to be very broad and rep- initiatives are a high priority with- resents ‘foundational’ patents in the in the USA and represent a large

712 DOI: 10.18609/cgti.2017.068 expert insight

ffFIGURE 2 Expansion of platelet-product markets.

Forward military theatres

Drug-delivery vehicle (US$50+B/year)

Platelet therapeutic product (US$15+B/year)

Cell culture media

Designer platelets (application dependent)

Radiation countermeasures

The number of potential markets for hiPSC-derived platelet products is rapidly expanding from its initial use as a therapeutic product into other market spaces including drug delivery, cell culture media, personalized medicine, and national security countermeasures. This trend of rapid market expansion will likely also apply to other areas of the regenerative medicine industry. potential market for hPSC-derived be fought in heavily populated ur- platelet products as a radiation ban centers. Due to the increasing countermeasure. Radiation expo- reach of missiles and the growing sure, such as would occur follow- risk of radiological attack at home, ing a nuclear accident or attack, allied countries’ platelet availability inhibits platelet production. As the is a national security issue. A large world grows smaller it is increas- radiological event would trigger an ingly likely that the next war will immediate demand for platelets

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that would deplete existing lo- used to transport drugs and hone cal inventory to treat emergency in on cancer cells by becoming en- trauma, followed by a sustained gineered to express antibodies [27], demand for platelets in survivors becoming tagged with antibodies 6+ days post-exposure. National on their surface [23], or being en- strategic stockpiles of platelets will gineered to express or cultured to become very important as our mil- ‘take-up’ various antibodies or mol- itary’s preparedness gap shifts from ecules into their secretory granules the front lines to the 24–48 hours [24,26]. Using these methods, plate- post-incident when affected popu- lets could be loaded with therapeu- lations become thrombocytopenic. tic molecules and directed to the de- The USA does not maintain an in- sired tissue (or tumor). The benefits ventory of platelets in the Strategic of this type of drug delivery include National Stockpile and there are no the ability to deliver molecules to licensed therapeutics that immedi- tissues that are traditionally hard to ately increase platelet counts. The target due to limitations imposed NIH Radiation Countermeasure by permeability or retention of the Program has specifically highlight- drug, and a reduced need to treat ed the Nation’s platelet supply as a patient systemically, which often a “critical unmet need” [66]. Our results in unwanted toxicities [69]. ability to bank platelet progeni- A third option is to create ‘design- tors and develop on-demand hiP- er platelets’ expressing desired char- SC-platelet production capabilities acteristics for targeted applications. will enable the establishment of a These tools can be leveraged to gen- strategic national stockpile of hiP- erate the specialized outcomes that SC-platelets that will be critical to personalized medicine approaches meeting this projected need. Along promise, without the drawbacks with rapid de novo production of that have prevented their commer- platelets following an attack, frozen cial implementation (high cost, pre-megakaryocyte intermediates time intensive, and inability to scale can be stored for extended periods products). Rather than generating of time and stockpiled to enable an custom hPSC lines from individual on-demanded rapid ‘surge’ produc- donors, it is preferential to develop tion of platelets when needed. a platform that utilizes cGMP-com- Another novel application of hP- pliant hPSC lines that are optimized SC-platelets is as a vehicle for drug for therapeutic product manufac- delivery. Platelets circulate in the ture. Genetic control of the hPSC bloodstream and touch every organ lines can then be applied to generate in the body, providing them with designer products for targeted thera- the potential to serve as part of a peutics and recipients. For example, versatile, customizable and targeta- designer platelets expressing high ble drug delivery system. Moreover, levels of clotting factors, e.g. Factor because of their immunomodulato- VII, in their granules could enhance ry and angiogenic functions, plate- clot formation at the site of damage lets are actively recruited by tumors without risk of systemic hypercoag- to aid in immune evasion and sup- ulation. These ‘hair-trigger platelets’ port their growth and metastasis could be targeted for trauma, in- [67,68]. Proof-of-concept studies creasing the effectiveness of platelet have shown that platelets can be transfusion during the first ‘Golden

714 DOI: 10.18609/cgti.2017.068 expert insight

Hour’ following severe traumatic in- ACKNOWLEDGEMENTS jury. Another potential application We would like to thank Jessica F. Olive for for designer platelets is in the treat- her insightful feedback and editing support. ment of fetal and neonatal alloim- We also acknowledge the generous funding mune thrombocytopenia (FNAIT). support of the NIH (#1R44HL131050-01, In this condition, fetal platelets ex- #1R43AI125134-01A1, and pressing a human platelet antigen #1SB1HL137591-01) and the (HPA) that their mother does not Massachusetts Life Sciences Center (MLSC express are targeted by the moth- Ramp Grant). er’s immune system, leading to fetal thrombocytopenia and serious po- FINANCIAL & COMPETING tential complications (including fetal INTERESTS DISCLOSURE intracranial hemorrhage) [70]. This condition could be treated using de- J.N.T. and S.M.K are partially supported signer platelets that have been engi- by NIH grants #1R44HL131050-01, neered with a single base pair change, #1R43AI125134-01A1, and such that HPA (negative) platelets #1SB1HL137591-01. J.N.T. and S.M.K. are administered to a HPA positive are paid employees of, have financial interest child after delivery by HPA-negative in, and are founders of Platelet BioGenesis, women, preventing the HPA-associ- a company that aims to produce donor- ated platelet clearance. independent human platelets from human- Other hPSC-derived therapeu- induced pluripotent stem cells at scale, and tics will undoubtedly discover sim- is an inventor on their patents. J.N.T. is an ilar second-generation opportuni- unpaid affiliate of Brigham and Women’s ties that build on the foundation Hospital and Harvard Medical School. The of their core technology, which is a interests of J.N.T. and S.M.K. were reviewed demonstration of the overall prom- and are managed by Platelet BioGenesis in ise of regenerative medicine. If ex- accordance with their conflict-of-interest ecuted properly, the returns from policies. these opportunities can far exceed the initial first generation product This work is licensed under envisioned by company founders, a Creative Commons Attri- both in terms of their ability to help bution – NonCommercial – NoDerivatives 4.0 patients and financially. To achieve International License this vision, we must first establish scalable, cGMP-compliant man- ufacturing processes that are safe, functional, and cost-competitive. The significant progress that has been made and that we, and others in the platelet space, continue to build upon is a vital model for other regenerative tissue markets to come.

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