CELL & GENE THERAPY INSIGHTS
STRATEGIES FOR SCALE-UP & SCALE-OUT
EXPERT INSIGHT Scale up of platelet production from human pluripotent stem cells for developing targeted therapies: advances and challenges
Jonathan N Thon & Sven M Karlsson
Without question, the future of regenerative medicine is in the scalable production of transfusable human tissues for therapeutic use. Platelets 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 blood type 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 cell culture, 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 granulocytes, the incidence of HLA antibodies follow- fact, there has been a historical pre- ing platelet transfusion 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 apheresis 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 Megakaryocyte 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 megakaryocytes 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.