Drug regulatory-compliant validation of a qPCR assay for bioanalysis studies of a cell therapy product with a special focus on matrix interferences in a wide range of organ tissues

Quantitative polymerase chain reaction (qPCR) has emerged as an important bioanalytical method for assessing the pharmacokinetics of human-cell-based medicinal products after xenotransplantation into immunodeficient mice. A particular challenge in bioanalytical qPCR studies is that the different tissues of the host organism can affect amplification efficiency and amplicon detection to varying degrees, and ignoring these matrix effects can easily cause a significant underestimation of the true number of target cells in a sample. Here, we describe the development and drug regulatory-compliant validation of a TaqMan qPCR assay for the quantification of mesenchymal stromal cells in the range of 125 to 20,000 cells/200 L lysate via the amplification of a human-specific, highly repetitive α-satellite DNA sequence of the chromosome 17 centromere region HSSATA17. An assessment of matrix effects in 14 different mouse tissues and blood revealed a wide range of spike recovery rates across the different tissue types, from 11 to 174%. Based on these observations, we propose performing systematic spike-and-recovery experiments during assay validation and correcting for the effects of the different tissue matrices on cell quantification in subsequent bioanalytical studies by multiplying the back-calculated cell number by tissue-specific factors derived from the inverse of the validated percent recovery rate

Process data of allogeneic ex vivo-expanded ABCB5+ mesenchymal stromal cells for human use: Off-the-shelf GMP-manufactured donor-independent ATMP

© 2020, The Author(s). Background: Human dermal mesenchymal stromal cells (MSCs) expressing the ATP-binding cassette (ABC) efflux transporter ABCB5 represent an easily accessible MSC population that, based on preclinical and first-in-human data, holds significant promise to treat a broad spectrum of conditions associated not only with skin-related but also systemic inflammatory and/or degenerative processes. Methods: We have developed a validated Good Manufacturing Practice-compliant expansion and manufacturing process by which ABCB5+ MSCs derived from surgical discard skin tissues are processed to an advanced-therapy medicinal product (ATMP) for clinical use. Enrichment for ABCB5+ MSCs is achieved in a three-step process involving plastic adherence selection, expansion in a highly efficient MSC-selecting medium, and immunomagnetic isolation of the ABCB5+ cells from the mixed culture. Results: Product Quality Review data covering 324 cell expansions, 728 ABCB5+ MSC isolations, 66 ABCB5+ MSC batches, and 85 final drug products reveal high process robustness and reproducible, reliable quality of the manufactured cell therapy product. Conclusion: We have successfully established an expansion and manufacturing process that enables the generation of homogenous ABCB5+ MSC populations of proven biological activity manufactured as a standardized, donor-independent, highly pure, and highly functional off-the-shelf available ATMP, which is currently tested in multiple clinical trials

Process development and safety evaluation of ABCB5+ limbal stem cells as advanced-therapy medicinal product to treat limbal stem cell deficiency

Background: While therapeutic success of the limbal tissue or cell transplantation to treat severe cases of limbal stem cell (LSC) deficiency (LSCD) strongly depends on the percentage of LSCs within the transplanted cells, prospective LSC enrichment has been hampered by the intranuclear localization of the previously reported LSC marker p63. The recent identification of the ATP-binding cassette transporter ABCB5 as a plasma membrane-spanning marker of LSCs that are capable of restoring the cornea and the development of an antibody directed against an extracellular loop of the ABCB5 molecule stimulated us to develop a novel treatment strategy based on the utilization of in vitro expanded allogeneic ABCB5+ LSCs derived from human cadaveric limbal tissue. Methods: We developed and validated a Good Manufacturing Practice- and European Pharmacopeia-conform production and quality-control process, by which ABCB5+ LSCs are derived from human corneal rims, expanded ex vivo, isolated as homogenous cell population, and manufactured as an advanced-therapy medicinal product (ATMP). This product was tested in a preclinical study program investigating the cells’ engraftment potential, biodistribution behavior, and safety. Results: ABCB5+ LSCs were reliably expanded and manufactured as an ATMP that contains comparably high percentages of cells expressing transcription factors critical for LSC stemness maintenance (p63) and corneal epithelial differentiation (PAX6). Preclinical studies confirmed local engraftment potential of the cells and gave no signals of toxicity and tumorgenicity. These findings were sufficient for the product to be approved by the German Paul Ehrlich Institute and the U.S. Food & Drug Administration to be tested in an international multicenter phase I/IIa clinical trial (NCT03549299) to evaluate the safety and therapeutic efficacy in patients with LSCD. Conclusion: Building upon these data in conjunction with the previously shown cornea-restoring capacity of human ABCB5+ LSCs in animal models of LSCD, we provide an advanced allogeneic LSC-based treatment strategy that shows promise for replenishment of the patient’s LSC pool, recreation of a functional barrier against invading conjunctival cells and restoration of a transparent, avascular cornea

Process development and safety evaluation of ABCB5(+) limbal stem cells as advanced-therapy medicinal product to treat limbal stem cell deficiency

Background While therapeutic success of the limbal tissue or cell transplantation to treat severe cases of limbal stem cell (LSC) deficiency (LSCD) strongly depends on the percentage of LSCs within the transplanted cells, prospective LSC enrichment has been hampered by the intranuclear localization of the previously reported LSC marker p63. The recent identification of the ATP-binding cassette transporter ABCB5 as a plasma membrane-spanning marker of LSCs that are capable of restoring the cornea and the development of an antibody directed against an extracellular loop of the ABCB5 molecule stimulated us to develop a novel treatment strategy based on the utilization of in vitro expanded allogeneic ABCB5(+) LSCs derived from human cadaveric limbal tissue. Methods We developed and validated a Good Manufacturing Practice- and European Pharmacopeia-conform production and quality-control process, by which ABCB5(+) LSCs are derived from human corneal rims, expanded ex vivo, isolated as homogenous cell population, and manufactured as an advanced-therapy medicinal product (ATMP). This product was tested in a preclinical study program investigating the cells' engraftment potential, biodistribution behavior, and safety. Results ABCB5(+) LSCs were reliably expanded and manufactured as an ATMP that contains comparably high percentages of cells expressing transcription factors critical for LSC stemness maintenance (p63) and corneal epithelial differentiation (PAX6). Preclinical studies confirmed local engraftment potential of the cells and gave no signals of toxicity and tumorgenicity. These findings were sufficient for the product to be approved by the German Paul Ehrlich Institute and the U.S. Food & Drug Administration to be tested in an international multicenter phase I/IIa clinical trial (NCT03549299) to evaluate the safety and therapeutic efficacy in patients with LSCD. Conclusion Building upon these data in conjunction with the previously shown cornea-restoring capacity of human ABCB5(+) LSCs in animal models of LSCD, we provide an advanced allogeneic LSC-based treatment strategy that shows promise for replenishment of the patient's LSC pool, recreation of a functional barrier against invading conjunctival cells and restoration of a transparent, avascular cornea

Deep Underground Neutrino Experiment (DUNE) Near Detector Conceptual Design Report

International audienceThe Deep Underground Neutrino Experiment (DUNE) is an international, world-class experiment aimed at exploring fundamental questions about the universe that are at the forefront of astrophysics and particle physics research. DUNE will study questions pertaining to the preponderance of matter over antimatter in the early universe, the dynamics of supernovae, the subtleties of neutrino interaction physics, and a number of beyond the Standard Model topics accessible in a powerful neutrino beam. A critical component of the DUNE physics program involves the study of changes in a powerful beam of neutrinos, i.e., neutrino oscillations, as the neutrinos propagate a long distance. The experiment consists of a near detector, sited close to the source of the beam, and a far detector, sited along the beam at a large distance. This document, the DUNE Near Detector Conceptual Design Report (CDR), describes the design of the DUNE near detector and the science program that drives the design and technology choices. The goals and requirements underlying the design, along with projected performance are given. It serves as a starting point for a more detailed design that will be described in future documents

The DUNE Far Detector Vertical Drift Technology, Technical Design Report

International audienceDUNE is an international experiment dedicated to addressing some of the questions at the forefront of particle physics and astrophysics, including the mystifying preponderance of matter over antimatter in the early universe. The dual-site experiment will employ an intense neutrino beam focused on a near and a far detector as it aims to determine the neutrino mass hierarchy and to make high-precision measurements of the PMNS matrix parameters, including the CP-violating phase. It will also stand ready to observe supernova neutrino bursts, and seeks to observe nucleon decay as a signature of a grand unified theory underlying the standard model. The DUNE far detector implements liquid argon time-projection chamber (LArTPC) technology, and combines the many tens-of-kiloton fiducial mass necessary for rare event searches with the sub-centimeter spatial resolution required to image those events with high precision. The addition of a photon detection system enhances physics capabilities for all DUNE physics drivers and opens prospects for further physics explorations. Given its size, the far detector will be implemented as a set of modules, with LArTPC designs that differ from one another as newer technologies arise. In the vertical drift LArTPC design, a horizontal cathode bisects the detector, creating two stacked drift volumes in which ionization charges drift towards anodes at either the top or bottom. The anodes are composed of perforated PCB layers with conductive strips, enabling reconstruction in 3D. Light-trap-style photon detection modules are placed both on the cryostat's side walls and on the central cathode where they are optically powered. This Technical Design Report describes in detail the technical implementations of each subsystem of this LArTPC that, together with the other far detector modules and the near detector, will enable DUNE to achieve its physics goals

The DUNE Far Detector Vertical Drift Technology, Technical Design Report

International audienceDUNE is an international experiment dedicated to addressing some of the questions at the forefront of particle physics and astrophysics, including the mystifying preponderance of matter over antimatter in the early universe. The dual-site experiment will employ an intense neutrino beam focused on a near and a far detector as it aims to determine the neutrino mass hierarchy and to make high-precision measurements of the PMNS matrix parameters, including the CP-violating phase. It will also stand ready to observe supernova neutrino bursts, and seeks to observe nucleon decay as a signature of a grand unified theory underlying the standard model. The DUNE far detector implements liquid argon time-projection chamber (LArTPC) technology, and combines the many tens-of-kiloton fiducial mass necessary for rare event searches with the sub-centimeter spatial resolution required to image those events with high precision. The addition of a photon detection system enhances physics capabilities for all DUNE physics drivers and opens prospects for further physics explorations. Given its size, the far detector will be implemented as a set of modules, with LArTPC designs that differ from one another as newer technologies arise. In the vertical drift LArTPC design, a horizontal cathode bisects the detector, creating two stacked drift volumes in which ionization charges drift towards anodes at either the top or bottom. The anodes are composed of perforated PCB layers with conductive strips, enabling reconstruction in 3D. Light-trap-style photon detection modules are placed both on the cryostat's side walls and on the central cathode where they are optically powered. This Technical Design Report describes in detail the technical implementations of each subsystem of this LArTPC that, together with the other far detector modules and the near detector, will enable DUNE to achieve its physics goals

Scintillation light detection in the 6-m drift-length ProtoDUNE Dual Phase liquid argon TPC

DUNE is a dual-site experiment for long-baseline neutrino oscillation studies, neutrino astrophysics and nucleon decay searches. ProtoDUNE Dual Phase (DP) is a 6 $\times$ 6 $\times$ 6 m$^3$ liquid argon time-projection-chamber (LArTPC) that recorded cosmic-muon data at the CERN Neutrino Platform in 2019–2020 as a prototype of the DUNE Far Detector. Charged particles propagating through the LArTPC produce ionization and scintillation light. The scintillation light signal in these detectors can provide the trigger for non-beam events. In addition, it adds precise timing capabilities and improves the calorimetry measurements. In ProtoDUNE-DP, scintillation and electroluminescence light produced by cosmic muons in the LArTPC is collected by photomultiplier tubes placed up to 7 m away from the ionizing track. In this paper, the ProtoDUNE-DP photon detection system performance is evaluated with a particular focus on the different wavelength shifters, such as PEN and TPB, and the use of Xe-doped LAr, considering its future use in giant LArTPCs. The scintillation light production and propagation processes are analyzed and a comparison of simulation to data is performed, improving understanding of the liquid argon properties.DUNE is a dual-site experiment for long-baseline neutrino oscillation studies, neutrino astrophysics and nucleon decay searches. ProtoDUNE Dual Phase (DP) is a 6x6x6m3 liquid argon time-projection-chamber (LArTPC) that recorded cosmic-muon data at the CERN Neutrino Platform in 2019-2020 as a prototype of the DUNE Far Detector. Charged particles propagating through the LArTPC produce ionization and scintillation light. The scintillation light signal in these detectors can provide the trigger for non-beam events. In addition, it adds precise timing capabilities and improves the calorimetry measurements. In ProtoDUNE-DP, scintillation and electroluminescence light produced by cosmic muons in the LArTPC is collected by photomultiplier tubes placed up to 7 m away from the ionizing track. In this paper, the ProtoDUNE-DP photon detection system performance is evaluated with a particular focus on the different wavelength shifters, such as PEN and TPB, and the use of Xe-doped LAr, considering its future use in giant LArTPCs. The scintillation light production and propagation processes are analyzed and a comparison of simulation to data is performed, improving understanding of the liquid argon properties

Reconstruction of interactions in the ProtoDUNE-SP detector with Pandora

International audienceThe Pandora Software Development Kit and algorithm libraries provide pattern-recognition logic essential to the reconstruction of particle interactions in liquid argon time projection chamber detectors. Pandora is the primary event reconstruction software used at ProtoDUNE-SP, a prototype for the Deep Underground Neutrino Experiment far detector. ProtoDUNE-SP, located at CERN, is exposed to a charged-particle test beam. This paper gives an overview of the Pandora reconstruction algorithms and how they have been tailored for use at ProtoDUNE-SP. In complex events with numerous cosmic-ray and beam background particles, the simulated reconstruction and identification efficiency for triggered test-beam particles is above 80% for the majority of particle type and beam momentum combinations. Specifically, simulated 1 GeV/$c$ charged pions and protons are correctly reconstructed and identified with efficiencies of 86.1$\pm0.6$% and 84.1$\pm0.6$%, respectively. The efficiencies measured for test-beam data are shown to be within 5% of those predicted by the simulation