Processes In Closed Systems Research Articles

The geochemistry of leucite-bearing lavas from early stages of the Somma-Vesuvius volcanic complex: Feeder systems and mantle enrichment processes in the Neapolitan district of the Roman Magmatic Province

The lavas of the Mt. Somma volcanic epoch were erupted during the early stage of the Somma-Vesuvius volcanic complex. These lavas are mildly differentiated with the presence of plagioclase-clinopyroxene-olivine- ± leucite-bearing rocks (leucite tephrites, leucite-bearing shoshonites, latites), also characterized by low in MgO, Cr and Ni, with a Sr-Nd-isotope range (87Sr/86Sr = 0.706865–0.707861; 143Nd/144Nd = 0.51244–0.51258) that overlaps with lavas of the late stage Vesuvius erupted after 1631 CE (late stage of the Somma-Vesuvius volcanic complex). Differentiation is dominated by closed-system processes, with fractional crystallization of clinopyroxene, calcic plagioclase, olivine, magnetite, and leucite. Open-system differentiation processes are subordinate and associated with limited interaction with crustal rocks. Oxygen isotopes on clinopyroxene and olivine phenocrysts (δ18O = 6.5–7.9 ‰) are higher than typical uncontaminated mantle magmas, suggesting a crustal contribution to the melt. Although open-system assimilation + fractional crystallization certainly took place, this process alone does not adequately reproduce the chemical and isotopic composition of the Mt. Somma ultrapotassic magmas. Therefore, a contribution from a recycled crustal component in the mantle source is required, but probably dominated by sediment-derived fluids and melts. The Mt. Somma lavas are characterized by distinctly different geochemical features compared to the mafic products of the neighboring volcanic areas (i.e., Phlegrean, Procida and Ischia volcanic fields), where the recycled crustal component is less pronounced.

Chemical Composition and Color of Short-Rotation Teak Wood Thermally Modified in Closed and Open Systems

Although the effect of thermal modification (TM) on teak wood color is well documented, few studies have been carried out on closed-system processes, and it remains unclear what the effect is of different processes on the same material. This work aimed to verify the effect of closed- and open-system processes of TM on the color of short-rotation teak wood. Thermally modified wood (TMW) was evaluated in a closed system at 160 °C (CS160) and in an open system at 185 °C and 210 °C (OS185 and OS210). We measured the moisture content (initial and final) of the wood and the corrected mass loss (CML). The chemical analyses encompassed the contents of alpha-cellulose, hemicelluloses, lignin, and extractives (total, in acetone and dichloromethane). Wood color was measured before and after TM according to the CIEL*a*b* color space. It was possible to achieve the same color using different processes of thermal modification (CS160 and OS210). TM reduced wood lightness (L*), red–green chromaticity coordinate (a*), and yellow–blue chromaticity coordinate (b*). L* and a* had the biggest and smallest variations, respectively. TMW color was significantly changed, even at the mildest condition tested (OS185, 0.33% CML).

Towards Automated Engineering of Donor-Derived T Lymphocytes into CRISPR/Cas9-Mediated CAR T Cells in a Closed-System

Chimeric antigen receptor (CAR)-expressing T cells have revolutionized immunotherapy of cancer. However, some patients derive no benefit from approved CARTcell therapies due to limitations such as toxicity and use of autologous cells which are sometimes unfit and risk manufacturing failures. Additional or more effective CARTcell therapies are needed. Harnessing the endogenous homology-directed DNA repair pathway via precision genome editing based on Cas9RNP/rAAV6, we and others have demonstrated the feasibility of targeting transgene(s) integration into specific gene site(s) including their incorporation under endogenous regulatory elements. Using this strategy, we aimed to engineer healthy donor-derived CAR T cells from αβ+ T lymphocytes. For enhanced safety, we metabolically reprogrammed the CAR T cells by inactivating the UMPS gene needed for uridine biosynthesis. The UMPS-/-CAR T cells become dependent on exogenous uridine thereby serving as a cell control mechanism. We hypothesized that donor-derived αβ+ T lymphocytes can be engineered using the Cas9RNP-rAAV6 strategy in an automated closed-system into controllable CAR T cells. We inactivated TRAC and UMPS gene loci in αβ+T lymphocytes using Lonza4D nucleofector. Electroporated cells were simultaneously transduced with CD19CAR-containing rAAV6 DNA vector. We obtained TRAC gene knockout up to 97% with knock-in up to 89%. Disruption of the UMPS gene would transform the cells to being auxotrophic and depend on uridine supply for viability and growth. To evaluate the CART cells' antitumor potency, we cocultured the CD19CART cells with GFP-expressing Nalm6 leukemia cell lines. The CD19CART cells eradicated the leukemia cells within 72 hours. To mimic relapse, we re-challenged cocultured CART with Nalm6 cells and the tumor cells were again depleted within 72 hours. Expectedly, IL-2 production was elevated in CD19CART cells challenged with Nalm6. Next, we tested the ability to control the cytotoxicity of the CD19CART cells by coculturing them with Nalm6 cells in separate groups with/without exogenous uridine. Interestingly, the CD19CART cells cocultured without uridine eradicated the target cells within 72 hours but lost their potency upon subsequent challenge (Figure 1). In contrast, CD19CAR T cells supplied with uridine remained potent and repeatedly eradicated target cells indicating the ability to turn off the potency of CART cells with uridine supply/withdrawal. To establish an automated closed-system for CART cells, we adopted the Miltenyi CliniMACS Prodigy with in-line electroporator (Elpo) for process engineering. First, we determined optimized parameters for electroporating αβ+ T lymphocytes on Elpo(Table1). Cells electroporated with these settings were (>80% viability) comparable to untreated cells. We first engineered CART cells with Elpo/Miltenyi reagents in an open system(i.e.cultivated Elpo-electroporated cells in culture vessels/incubators). We obtained 95% of TRAC and UMPS gene knockouts and up to 90% CD19CAR transgene knock-in to the TRAC locus - an efficiency slightly higher than using Lonza 4D nucleofector. To scale up the Cas9-based T cell engineering into a single automated process on the CliniMACS Prodigy, the Cas9 and guide RNA reagents would be scaled up 1000fold. To make the process cost-effective, we tested AZD7468, a small molecule DNAPKcs inhibitor. We optimized/reduced the amounts of reagents upto 90% less of Cas9RNP used without any significant reduction in gene editing outcomes. AZD7648 reduced the MOI needed for viral transduction by 50% while maintaining 80% or higher levels of transgene knock-in efficiency. Next, we commenced pilot studies to automate the CAR T cell engineering process in closed-system. Currently, we have achieved an automated TRAC gene knockout of over 90% and CAR transgene knock-in of slightly >10%. Further optimization studies are underway to improve automated gene knock-in efficiency. We have engineered donor-derived αβ+ T lymphocytes into potent/controllable CD19CART cells. The UMPS-deficient CD19CART cells lose antileukemia activity without uridine. We show that metabolic reprogramming can be combined with precision genome editing to generate controllable CART cells. Finally, our data demonstrate the feasibility of automating CRISPR/Cas9-mediated engineering of CAR T cells within closed-system.

The formation and aqueous alteration of CM2 chondrites and their relationship to CO3 chondrites: A fresh isotopic (O, Cd, Cr, Si, Te, Ti, and Zn) perspective from the Winchcombe CM2 fall

AbstractAs part of an integrated consortium study, we have undertaken O, Cd, Cr, Si, Te, Ti, and Zn whole rock isotopic measurements of the Winchcombe CM2 meteorite. δ66Zn values determined for two Winchcombe aliquots are +0.29 ± 0.05‰ (2SD) and +0.45 ± 0.05‰ (2SD). The difference between these analyses likely reflects sample heterogeneity. Zn isotope compositions for Winchcombe show excellent agreement with published CM2 data. δ114Cd for a single Winchcombe aliquot is +0.29 ± 0.04‰ (2SD), which is close to a previous result for Murchison. δ130Te values for three aliquots gave indistinguishable results, with a mean value of +0.62 ± 0.01‰ (2SD) and are essentially identical to published values for CM2s. ε53Cr and ε54Cr for Winchcombe are 0.319 ± 0.029 (2SE) and 0.775 ± 0.067 (2SE), respectively. Based on its Cr isotopic composition, Winchcombe plots close to other CM2 chondrites. ε50Ti and ε46Ti values for Winchcombe are 3.21 ± 0.09 (2SE) and 0.46 ± 0.08 (2SE), respectively, and are in line with recently published data for CM2s. The δ30Si composition of Winchcombe is −0.50 ± 0.06‰ (2SD, n = 11) and is essentially indistinguishable from measurements obtained on other CM2 chondrites. In conformity with petrographic observations, oxygen isotope analyses of both bulk and micromilled fractions from Winchcombe clearly demonstrate that its parent body experienced extensive aqueous alteration. The style of alteration exhibited by Winchcombe is consistent with relatively closed system processes. Analysis of different fractions within Winchcombe broadly support the view that, while different lithologies within an individual CM2 meteorite can be highly variable, each meteorite is characterized by a predominant alteration type. Mixing of different lithologies within a regolith environment to form cataclastic matrix is supported by oxygen isotope analysis of micromilled fractions from Winchcombe. Previously unpublished bulk oxygen isotope data for 12 CM2 chondrites, when combined with published data, define a well‐constrained regression line with a slope of 0.77. Winchcombe analyses define a more limited linear trend at the isotopically heavy, more aqueously altered, end of the slope 0.77 CM2 array. The CM2 slope 0.77 array intersects the oxygen isotope field of CO3 falls, indicating that the unaltered precursor material to the CMs was essentially identical in oxygen isotope composition to the CO3 falls. Our data are consistent with earlier suggestions that the main differences between the CO3s and CM2s reflect differing amounts of water ice that co‐accreted into their respective parent bodies, being high in the case of CM2s and low in the case of CO3s. The small difference in Si isotope compositions between the CM and CO meteorites can be explained by different proportions of matrix versus refractory silicates. CMs and COs may also be indistinguishable with respect to Ti and Cr isotopes; however, further analysis is required to test this possibility. The close relationship between CO3 and CM2 chondrites revealed by our data supports the emerging view that the snow line within protoplanetary disks marks an important zone of planetesimal accretion.

Optimizing a fully automated and closed system process for red blood cell reduction of human bone marrow products

Background aimsHematopoietic stem cell transplantation using bone marrow as the graft source is a common treatment for hematopoietic malignancies and disorders. For allogeneic transplants, processing of bone marrow requires the depletion of ABO-mismatched red blood cells (RBCs) to avoid transfusion reactions. Here the authors tested the use of an automated closed system for depleting RBCs from bone marrow and compared the results to a semi-automated platform that is more commonly used in transplant centers today. The authors found that fully automated processing using the Sepax instrument (Cytiva, Marlborough, MA, USA) resulted in depletion of RBCs and total mononuclear cell recovery that were comparable to that achieved with the COBE 2991 (Terumo BCT, Lakewood, CO, USA) semi-automated process. MethodsThe authors optimized the fully automated and closed Sepax SmartRedux (Cytiva) protocol. Three reduction folds (10×, 12× and 15×) were tested on the Sepax. Each run was compared with the standard processing performed in the authors’ center on the COBE 2991. Given that bone marrow is difficult to acquire for these purposes, the authors opted to create a surrogate that is more easily obtainable, which consisted of cryopreserved peripheral blood stem cells that were thawed and mixed with RBCs and supplemented with Plasma-Lyte A (Baxter, Deerfield, IL, USA) and 4% human serum albumin (Baxalta, Westlake Village, CA, USA). This “bone marrow-like” product was split into two starting products of approximately 600 mL, and these were loaded onto the COBE and Sepax for direct comparison testing. Samples were taken from the final products for cell counts and flow cytometry. The authors also tested a 10× Sepax reduction using human bone marrow supplemented with human liquid plasma and RBCs. ResultsRBC reduction increased as the Sepax reduction rate increased, with an average of 86.06% (range of 70.85–96.39%) in the 10×, 98.80% (range of 98.1–99.5%) in the 12× and 98.89% (range of 98.80–98.89%) in the 15×. The reduction rate on the COBE ranged an average of 69.0–93.15%. However, white blood cell (WBC) recovery decreased as the Sepax reduction rate increased, with an average of 47.65% (range of 38.9–62.35%) in the 10×, 14.56% (range of 14.34–14.78%) in the 12× and 27.97% (range of 24.7–31.23%) in the 15×. COBE WBC recovery ranged an average of 53.17–76.12%. Testing a supplemented human bone marrow sample using a 10× Sepax reduction resulted in an average RBC reduction of 84.22% (range of 84.0–84.36%) and WBC recovery of 43.37% (range of 37.48–49.26%). Flow cytometry analysis also showed that 10× Sepax reduction resulted in higher purity and better recovery of CD34+, CD3+ and CD19+ cells compared with 12× and 15× reduction. Therefore, a 10× reduction rate was selected for the Sepax process. ConclusionsThe fully automated and closed SmartRedux program on the Sepax was shown to be effective at reducing RBCs from “bone marrow-like” products and a supplemented bone marrow product using a 10× reduction rate.

Closed system processing variables affect post-thaw quality characteristics of cryopreserved red cell concentrates.

Differences in manufacturing conditions using the Haemonetics ACP 215 cell processor result in cryopreserved red cell concentrates (RCCs) of varying quality. This work studied the effect of processing method, additive solution, and storage duration on RCC quality to identify an optimal protocol for the manufacture of cryopreserved RCCs. RCCs were pooled-and-split and stored for 7, 14, or 21 days before cryopreservation. Units were glycerolized with the ACP 215 using a single or double centrifugation method. After thawing, the RCCs were deglycerolized, suspended in AS-3, SAGM, ESOL, or SOLX/AS-7, and stored for 0, 3, 7, 14, or 21 days before quality testing. Quality assessments included hemoglobin content, hematocrit, hemolysis, adenosine triphosphate (ATP), supernatant potassium, and mean cell volume. Both glycerolization methods produced RCCs that met regulatory standards for blood quality. Dual centrifugation resulted in higher hemoglobin content, fewer processing alerts, and a shorter deglycerolization time than single centrifugation processing. Units processed with AS-3 and ESOL met regulatory standards when stored for up to 21 days pre-cryopreservation and 21 days post-deglycerolization. However, ESOL demonstrated superior maintenance of ATP over RBCs in AS-3. Some RCCs suspended in SAGM and SOLX exceeded acceptable hemolysis values after 7 days of post-deglycerolization storage regardless of pre-processing storage length. When manufacturing cryopreserved RCCs using the ACP 215, dual centrifugation processing with AS-3 or ESOL additive solutions is preferred, with storage periods of up to 21 days both pre-processing and post-deglycerolization.

Possibilities for continuous closed-system processing of cell therapies

Cell and gene therapies have the potential to facilitate disease-modifying treatment of both rare and chronic conditions. Whilst approved treatments are now available, several challenges remain before these therapeutic modalities become first in line therapies. The supply and qualification of starting materials such as peripheral blood and bone marrow is one dilemma that must be overcome. For allogeneic programs, starting materials generally have a defined limit of productivity and carry requirements of substantial risk-mitigation testing. Autologous manufacturing processes, with lower attendant safety risks, are highly variable and may be compromised based on individual patient treatment programs or the target disease itself. An ideal starting material has a high capacity for expansion, providing consistent manufacturing, reduced qualification burden, and a high output of individual doses. However, even if a developer is working with an ideal starting material, manufacturing processes may limit the ability to capitalize on beneficial characteristics. One possible solution to improving manufacturing capacity is incorporation of continuous batch manufacturing.

Crustal melting and suprasolidus phase equilibria: From first principles to the state-of-the-art

Partial melting is the fundamental process by which juvenile crust was produced from the mantle and subsequently reworked to become the stable, compositionally-differentiated continents on which we live and which host most of the elemental resources required by our modern technological society. Irreversible differentiation of the continental crust occurs principally through the production, segregation and migration of silica-rich (felsic) melts from deeper source rocks to shallower sinks where they erupt or, more commonly, crystallise as granite sensu lato. Here we provide for both novices and professionals a comprehensive but accessible account of the processes involved in crustal melting and suprasolidus phase equilibria from first principles to the forefront of modern research. To begin, we introduce the tectono-metamorphic context for crustal melting before considering the evidence at outcrop and in thin section for partial melting, then briefly review melt extraction from crustal rocks. As a prerequisite to understanding the physicochemical basis for crustal melting, we summarize the essential thermodynamics that underpin calculated phase equilibria, distinguish different types of melting reaction, and review the requirements for, methodology behind, and limitations of a phase equilibrium modelling approach based on equilibrium thermodynamics. We explain the various types of phase diagram used to investigate partial melting and assess open versus closed system processes, including internal and external buffering of H2O. Those crustal sources that partially melt to produce granite are considered in detail, namely basic rocks such as basalts and gabbros, clastic sedimentary rocks such as greywackes, siltstones and mudstones (pelites), and granites themselves. We concentrate mainly on intracrustal partial melting in convergent-margin settings, and on anatexis during exhumation of deeply-subducted continental crust from mantle depths. We discuss the behaviour of trace elements and accessory minerals during melting, and consider the implications for isotope geochemistry. To close we include a brief summary of some of the important points and offer a few suggestions for future lines of research on crustal melting.

Petrological characterization of the Cenozoic igneous rocks of the Tafresh district, central Urumieh-Dokhtar Magmatic Arc (Iran)

We report a petrographic and whole-rock geochemical characterization of the Cenozoic volcanic rocks cropping out in the Tafresh area of the central Urumieh-Dokhtar Magmatic Arc of Iran. The investigated rocks range mainly from basaltic andesite to dacite, and are considered to be genetically linked by (mostly) closed-system evolutionary processes involving fractionation of ferromagnesian minerals and plagioclase first, then of plagioclase and lesser amphibole (plus minor clinopyroxene) and finally of plagioclase with lesser alkali feldspar and minor amphibole. These represent a typical calcalkaline series emplaced in a subduction-related setting, producing the observed LILE-enriched and HFSE-depleted geochemical signature. The basaltic andesite compositions likely derived from an unsampled hydrous primitive melt equilibrated in a spinel-bearing metasomatized peridotite source, evolving at shallow to moderate crustal depths.

Deep and disturbed: conditions for formation and eruption of a mingled rhyolite at Ascension Island, south Atlantic

The generation of felsic melts (through open or closed system processes) within ocean island volcanoes has been a key area of study since their identification. At Ascension Island in the south Atlantic, explosively erupted felsic melts have, to date, demonstrated a marked absence of signs of magma mixing and crustal assimilation. Here we present the first observations of a fall deposit from Ascension Island recording both macro- and micro-scale evidence for magma mingling. Geochemical analyses of mineral and glass phases, coupled with volatile concentrations of melt inclusions highlight the role of lower-crustal partial melting to produce rhyolitic magmas. Glass textures and the lack of zoning in major mineral phases indicate that mingling with a mafic melt occurred shortly prior to eruption. These inferences of a deep rhyolite production zone, coupled with rapid ascent rates highlight the challenges in forecasting a similar style of eruption at Ascension Island in the future.

Development of a closed system process for purifying naive CD8+ cells, culturing and transducing with a CD19/22 chimeric antigen receptor (CAR) to produce a clinical T memory stem cell product directed against B cell malignancies

Background & Aim Currently in the Center for Cellular Engineering at the National Institutes of Health, a clinical trial which involves purifying CD8+CD62L+CD45RA+ naive T cells, culturing and transducing with a retroviral CD19 CAR, is underway. Mononuclear cells are enriched from apheresis product by automated density gradient, followed by 3 separate Strep-tactin® magnetic microbead manual selections. The selected Naive T cells are then transduced and cultured for 7 days in gas permeable cell culture bags. The entire process is open, especially purification procedures which are extremely labor intensive, takes between 16-18 hours and is performed over two days. Therefore, a less labor intensive, more streamlined and closed system process is needed. Methods, Results & Conclusion We developed a more efficient and GMP compliant process for generating CD19 and CD22 CAR Tscm product. A large scale CD8+ selection was performed on the CliniMacs Prodigy® platform followed by sterile, closed system cell sorting on the MACSQuant Tyto® for the purification of naive T cells (targets). These purified cells were placed back on the the Prodigy for culture and transduction using an automated customized program. While these enrichments worked well for donors with relatively normal target percentages, several older donors with low targets did not reach the desired purity. We then adopted a CD8+ releasable microbead selection, performed on the CliniMacs Plus device, followed by a CD62L+ microbead selection (Miltenyi Biotec) on the CliniMacs Prodigy to further enrich for target cells. We have performed many small scale selections and sorts and can consistently achieve 80-90% purities utilizing two selections. New MACSQuant Tyto® HS cartridges are now available which could potentially cut the sorting time in half, and along with new recently developed software program to decrease user setting manipulations, resulting in decreased sorting times and increased purities. In addition, after optimizing culture conditions in the CliniMacs Prodigy, as few as 25 million naive T cells can be initiated for culture, and a naive phenotype can be retained after 7 days in culture with a single lentiviral transduction. In summary, we developed a robust GMP compliant process for Tscm purification, culture and transduction which can be used to generate CAR Tscm products.

Human natural killer cell manufacturing using a closed system process for patients with metastatic solid tumors or hematologic malignancies

Background & Aim As potent effectors of the innate immune system natural killer (NK) cells are currently utilized in clinical trials world-wide to treat a variety of hematologic and solid organ malignancies. The ongoing phase I/II study at the National Institutes of Health (08-H-0186) previously utilized open-system processing requiring 60-80 small GREX flasks and single donor AB plasma in the culture medium. We optimized the manufacturing process to generate large numbers of NK cells using closed-system processing with large 5 liter GREX containers and commercially available pooled AB serum. Methods, Results & Conclusion The manufacturing process entails two stages: Preparation of the feeder EBV-transformed lymphoblastoid cell line (LCL) and NK cell culture. LCL are cultured in closed GREX100-CS vessels with XVIVO15 culture media containing 15mM HEPES, 2mM glutamax and 10% of either commercially available pooled AB serum or single donor AB plasma. With one culture vessel capable of generating 8-10 × 108 LCL feeder cells using 600-800mL of culture medium, 3 vessels sufficiently support a full scale culture. LCL number and viability are similar between groups containing serum vs. plasma. In the second stage, NK cell culture is initiated using 3 GREX500-CS with 40 × 106 NK cells and 400 × 106 irradiated LCL feeder cells in 500mL/vessel. Fresh XVIVO20 containing 15mM HEPES, 2mM glutamax, 500IU/mL IL2 and either 10% AB plasma or pooled AB serum is added on days 4, 8, 12 and 17. On day 14, a portion of the cells are harvested and washed for fresh infusion, and the remaining cells are re-seeded into a new vessel for harvest for second infusion on day 19. Day 14 and day 19 harvest generated 10-14 × 109 (1 vessel) and 50-70 × 109 NK cells (4-5 vessels) for fresh infusion, enough to support the target dose level of 1 × 108 (1st harvest) and 5 × 108 (2nd harvest) NK cells/kg. We found no significant differences in cell growth, viability or TRAIL expression when using AB plasma vs. AB serum. However, TRAIL expression was significantly increased when the concentration of the NK cells was kept below 4 × 106 cells/mL during culture. NK cells also demonstrated tumor cell line killing ability in vitro and had increased CD107. In summary, we have developed a new process that improves safety and consistency for culturing NK cells in large numbers for an ongoing clinical trial at the NIH. This process can be easily adapted by cell therapy manufacturing facilities at most academic institutions.

Automated closed-system large-scale expansion of clinical-grade natural killer cells

Background & Aim The field of cellular immunotherapy is growing rapidly. Natural killer (NK) cells are part of the innate immune system and have wide potential for therapeutic application. Although CAR-T cells are now commercially available, NK cells are still in pre-commercial development and testing. It is likely that much higher numbers of NK cells will be required to achieve therapeutic results compared to that of antigen-specific T cells. With the development of feeder cells expressing membrane-bound IL-21, it has become possible to achieve large-scale expansion of non-senescent NK cells. To date, most studies have expanded NK cells at clinical scale by sequential application of open systems (G-rex) or small-volume closed systems. Achieving large-scale expansion of GMP-grade NK cells requires multiple systems operating in parallel, which increase labor costs, material costs and potential for contamination. To develop a large-scale process for NK-cell expansion in compliance with regulatory requirements, we combined the CliniMACS Prodigy (Miltenyi Biotec) and Xuri W25 (GE Healthcare) into one closed-system process. Methods, Results & Conclusion In collaboration with Miltenyi, we optimized a custom program for the Prodigy to perform Ficoll density-gradient selection of mononuclear cells, CD3-based magnetic depletion of T cells, periodic addition of and forced interaction with feeder cells, and on-demand addition of media for the first phase of NK-cell culture, culminating in transfer of the cells to a secondary culture bag. We then applied a semi-continuous perfusion program on the Xuri for the second phase. We identified optimal glucose and pH levels to support log-phase expansion, and refined the Prodigy program to optimize CD3-depletion and allow efficient volume reduction steps for media exchanges. On the Xuri, pH and dissolved oxygen (DO) monitoring was utilized to identify optimal rocking rates, rocking angles and perfusion parameters to allow sufficient NK cell-feeder cell contact and achieve high cell densities. Finally, NK cells produced by this approach demonstrated very high cytotoxicity (mean 65.2% at 1.25:1 E:T ratio). With these optimized protocols, we achieved high-purity (≥99%, n=3) and high-density (∼107 cells/mL) conditions to yield 5 × 1010 high-activity NK cells in a 5L final culture volume from one donor buffy coat. We anticipate realizing both reduced cost and increased regulatory compliance of NK cell production by using the automated closed systems of Prodigy and Xuri in sequence.

The Rorschach Omnipotence Scale and closed-system processing.

The Rorschach Omnipotence Scale and closed-system processing.

Supplemental Material for The Rorschach Omnipotence Scale and Closed-System Processing

Supplemental Material for The Rorschach Omnipotence Scale and Closed-System Processing

Petrogenesis of peralkaline granite dykes of the Straumsvola complex, western Dronning Maud Land, Antarctica

Peralkaline syenite and granite dykes cut the Straumsvola nepheline syenite pluton in Western Dronning Maud Land, Antarctica. The average peralkalinity index (PI = molecular Al/[Na + K]) of the dykes is 1.20 (n = 29) and manifests itself in the presence of the Zr silicates eudialyte, dalyite and vlasovite, and the Na–Ti silicate, narsarsukite. The dykes appear to have intruded during slow cooling of the nepheline syenite pluton, and the petrogenetic relationship of the dykes and the pluton cannot be related to closed-system processes at low pressure, given the thermal divide that exists between silica-undersaturated and oversaturated magmas. Major and trace element variations in the dykes are consistent with a combination of fractional crystallization of parental peralkaline magma of quartz trachyte composition, and internal mineral segregation prior to final solidification. The distribution of accessory minerals is consistent with late-stage crystallization of isolated melt pockets. The dykes give an Rb–Sr isochron age of 171 ± 4.4 Ma, with variable initial 87Sr/86Sr ratio (0.7075 ± 0.0032), and have an average e Nd of − 12.0. Quartz phenocrysts have δ18O values of 8.4–9.2‰, which are generally in O-isotope equilibrium with bulk rock. Differences in the δ18O values of quartz and aegirine (average Δquartz−aegirine = 3.5‰) suggest aegirine formation temperatures around 500 °C, lower than expected for a felsic magma, but consistent with poikilitic aegirine that indicates subsolidus growth. The negative e Nd (< − 10) and magma δ18O values averaging 8.6‰ (assuming Δquartz−magma = 0.6‰) are inconsistent with a magma produced by closed-system fractional crystallization of a mantle-derived magma. By contrast, the nepheline syenite magma had mantle-like δ18O values and much less negative e Nd (average − 3.1, n = 3). The country rock has similar δ18O values to the granite dykes (average 8.0‰, n = 108); this means that models for the petrogenesis of the granites by assimilation are unfeasible, unless an unexposed high-δ18O contaminant is invoked. Instead, it is proposed that the peralkaline syenite and granite dykes formed by partial melting of alkali-metasomatised gneiss that surrounds the nepheline syenite, followed by fractional crystallization.

Closed-system manufacturing of CD19 and dual-targeted CD20/19 chimeric antigen receptor T cells using the CliniMACS Prodigy device at an academic medical center

Background aimsMultiple steps are required to produce chimeric antigen receptor (CAR)-T cells, involving subset enrichment or depletion, activation, gene transduction and expansion. Open processing steps that increase risk of contamination and production failure are required. This complex process requires skilled personnel and costly clean-room facilities and infrastructure. Simplified, reproducible CAR-T-cell manufacturing with reduced labor intensity within a closed-system is highly desirable for increased availability for patients. MethodsThe CliniMACS Prodigy with TCT process software and the TS520 tubing set that allows closed-system processing for cell enrichment, transduction, washing and expansion was used. We used MACS-CD4 and CD8-MicroBeads for enrichment, TransAct CD3/CD28 reagent for activation, lentiviral CD8 TM-41BB-CD3 ζ-cfrag vectors expressing scFv for CD19 or CD20/CD19 antigens for transduction, TexMACS medium-3%-HS-IL2 for culture and phosphate-buffered saline/ethylenediaminetetraacetic acid buffer for washing. Processing time was 13 days. ResultsEnrichment (N = 7) resulted in CD4/CD8 purity of 98 ± 4.0%, 55 ± 6% recovery and CD3+ T-cell purity of 89 ± 10%. Vectors at multiplicity of infection 5–10 resulted in transduction averaging 37%. An average 30-fold expansion of 108 CD4/CD8-enriched cells resulted in sufficient transduced T cells for clinical use. CAR-T cells were 82–100% CD3+ with a mix of CD4+ and CD8+ cells that primarily expressed an effector-memory or central-memory phenotype. Functional testing demonstrated recognition of B-cells and for the CAR-20/19 T cells, CD19 and CD20 single transfectants were recognized in cytotoxic T lymphocyte and interferon-γ production assays. DiscussionThe CliniMACS Prodigy device, tubing set TS520 and TCT software allow CAR-T cells to be manufactured in a closed system at the treatment site without need for clean-room facilities and related infrastructure.

Slab-derived halogens and noble gases illuminate closed system processes controlling volatile element transport into the mantle wedge

Halogen and noble gas systematics are powerful tracers of volatile recycling in subduction zones. We present halogen and noble gas compositions of mantle peridotites containing H2O-rich fluid inclusions collected at volcanic fronts from two contrasting subduction zones (the Avacha volcano of Kamchatka arc and the Pinatubo volcano of Luzon arcs) and orogenic peridotites from a peridotite massif (the Horoman massif, Hokkaido, Japan) which represents an exhumed portion of the mantle wedge. The aims are to determine how volatiles are carried into the mantle wedge and how the subducted fluids modify halogen and noble gas compositions in the mantle. The halogen and noble gas signatures in the H2O-rich fluids are similar to those of marine sedimentary pore fluids and forearc and seafloor serpentinites. This suggests that marine pore fluids in deep-sea sediments are carried by serpentine and supplied to the mantle wedge, preserving their original halogen and noble gas compositions. We suggest that the sedimentary pore fluid-derived water is incorporated into serpentine through hydration in a closed system along faults at the outer rise of the oceanic, preserving Cl/H2O and 36Ar/H2O values of sedimentary pore fluids. Dehydration–hydration process within the oceanic lithospheric mantle maintains the closed system until the final stage of serpentine dehydration. The sedimentary pore fluid-like halogen and noble gas signatures in fluids released at the final stage of serpentine dehydration are preserved due to highly channelized flow, whereas the original Cl/H2O and 36Ar/H2O ratios are fractionated by the higher incompatibility of halogens and noble gases in hydrous minerals.

Process automation in manufacturing of mesenchymal stromal cells.

Process automation in manufacturing of mesenchymal stromal cells.

BPOG Response to Annex 2.

This paper is an interpretive response to Annex 2 of Eudralex—Volume 4; the European Union (EU) guidelines for good manufacturing practice (GMP) of medicinal products for human and veterinary use ([1][1]). It was written collaboratively, by a team of experts in closed system processing, from 26