Process and analytical development challenges for the incorporation of gene edits into T cell therapies

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Gold surface with gold nitride–a surface enhanced Raman scattering active substrate

The nitration of gold surfaces is a nonpolluting method, which can lead to large scale production of substrates with remarkable properties and applications. We present a topographical study of the nanoscale structure of the gold nitride surfaces produced by radio frequency (rf) nitrogen plasma etching of thin gold films. Atomic force microscopy images taken after rf etching reveal the striking appearance of the cluster assembly with large clusters surrounded by small clusters (7.9±1.4 and 2.3±0.9 nm, respectively) appearing to exhibit an attractive interaction. We discuss the possible mechanism for this attraction based on a colloid model by Messina et al. [Phys. Rev. Lett. 85, 872 (2000) ]. This surface exhibits a notable surface enhanced Raman scattering effect demonstrated with L-alanine and rhodamine-6G. The significance of this work is that we found that this SERS active gold nitride surface can be prepared in just one step: by nitrogen plasma etching a thin gold film. Until now most SERS active gold cluster covered surfaces have been prepared in several steps very often requiring complex lithography

Scale-down and initial characterization studies of an allogeneic cell therapy manufacturing process

Cancer therapies leveraging the immune system have been improving the course of disease. T-cell therapies are one of the modalities of immune therapy, which also include small molecules, proteins, and various types of immune cells. T-cell therapies include T-cell Receptor cells (TCR), Chimeric Antigen Receptor (CAR) T-cells, and Cytotoxic T-Lymphocytes (CTL). Although the nascent cell therapy industry faces many manufacturing challenges, it benefits from the vast experience of the blood industry and cell-produced biologics industry. A notable manufacturing challenge for most T-cell therapies results from their autologous nature, in which cells from a patient are used to produce the therapeutic cells for the same patient. The resultant patient-specific manufacture introduces new challenges to scale-out throughout the process, particularly for cell culture. In common with classic therapeutic protein scale-out approaches, a large number of small-scale cultures must be run simultaneously at the commercial stage. However, patient-specific cell therapies require that each culture is performed using cells from the patient to be treated and is adequately separated from others to prevent cross-contamination. Further, the cultures behave differently for each patient due to the differences in the starting patient-derived cell seed. Finally, T-cell growth is typically induced by an activation step (via specific antigens or non-specific activation signaling). Accordingly, a culture stage allowing undisturbed cellular interactions at the initiation of T-cell cultures is required. A number of approaches are available to address the challenges of cost-effective culture scale-out for T-cell therapies. These approaches include GMP culture platforms from the therapeutic protein and blood industries. Platforms using disposable cell contact surfaces are predominantly favored to minimize the risk of cross-contamination and to provide rapid turnover between patient-batches. Rocking platform bioreactors such as the WAVE have been extensively used for T-cell cultures due to simplicity and ease of closed-system manipulations. However, the need for undisturbed cell-cell interactions to achieve T-cell activation early in the culture demands static seed trains in another platform. Static cultures based on gas-permeable vessels that require minimal operator preparation are also widely used. One example is gas-permeable culture bags filled using closed-system weld connections. Another example is the Gas-permeable Rapid Expansion (G-Rex) device, a static culture vessel in which the cells rest on a gas permeable surface. In this device, excellent membrane permeability allows high densities of cells to grow on the bottom of the vessel. Further, the passive gas exchange at the bottom of the vessel allows large amounts of medium to be placed into the vessel without compromising gas exchange. This allows long-term cultures with little need for medium exchange, if any. We compare cultures in G-Rex, gas-permeable bags, and the WAVE bioreactor to demonstrate that all produce sufficient cell expansion with appropriate cell characteristics. However, some T-cell immunotherapies may have particular culture needs that favor one platform over the others. In addition, we observe activation in static cultures and characterize the undisturbed time necessary. We illustrate examples of scale-out for each culture platform and discuss consequences. Finally, we generate models to compare projected commercial costs of the culture platforms. Together, our studies illustrate three disposable culture systems that have the potential to enable commercialization of multiple types of T-cell therapies

A western boundary current eddy characterisation study

The analysis of an eddy census for the East Australian Current (EAC) region yielded a total of 497 individual short-lived (7-28 days) cyclonic and anticyclonic eddies for the period 1993 to 2015. This was an average of about 23 eddies per year. 41% of the tracked individual cyclonic and anticyclonic eddies were detected off southeast Queensland between about 25 oS and 29 oS. This is the region where the flow of the EAC intensifies forming a swift western boundary current that impinges near Fraser Island on the continental shelf. This zone was also identified as having a maximum in detected short-lived cyclonic eddies. A total of 94 (43%) individual cyclonic eddies or about 4-5 per year were tracked in this region. The census found that these potentially displaced entrained water by about 115 km with an average displacement speed of about 4 km per day. Cyclonic eddies were likely to contribute to establishing an on-shelf longshore northerly flow forming the western branch of the Fraser Island Gyre and possibly presented an important cross-shelf transport process in the life cycle of temperate fish species of the EAC domain. In-situ observations near western boundary currents previously documented the entrainment, off-shelf transport and export of near shore water, nutrients, sediments, fish larvae and the renewal of inner shelf water due to short-lived eddies. This study found that these cyclonic eddies potentially play an important off-shelf transport process off the central east Australian coast

Is the East Australian Current causing a marine ecological hot-spot and an important fisheries near Fraser Island, Australia?

The distributions of chlorophyll-a (Chl-a) blooms near the Fraser Island continental shelf along the east coast of Australia were analysed for the period 2002–2012. The blooms were found to exhibit two distinct quasi climatological patterns. The first pattern was a broad near-coast mid-shelf distribution that prevailed from about March to July each year. The second pattern was established due to re-occurring outer-shelf Chl-a blooms southeast of Fraser Island from about August to February. The outer-shelf Chl-a bloom concentration maxima appeared to be higher than those associated with the near coast pattern. Both distributions were found to be characterised by significant year-to-year variability in the number of total blooms, the length of blooms and the Chl-a bloom concentration maxima. The physical cause of the outer-shelf Chl-a concentration maxima was of particular interest, since this location overlaps with a region previously identified as a key eastern Australian marine ecological site and important fisheries. In this analysis, we found that the area also overlaps with a hot-spot in EAC-generated bottom layer stress, which appears to be the main driver of the 'Southeast Fraser Island Upwelling System'

A Novel Artificial Organic Controller with Hermite Optical Flow Feedback for Mobile Robot Navigation

This chapter describes a novel nature-inspired and intelligent control system for mobile robot navigation using a fuzzy-molecular inference (FMI) system as the control strategy and a single vision-based sensor device, that is, image acquisition system, as feedback. In particular, FMI system is proposed as a hybrid fuzzy inference system with an artificial hydrocarbon network structure as defuzzifier that deals with uncertainty in motion feedback, improving robot navigation in dynamic environments. Additionally, the robotics system uses processed information from an image acquisition device using a real-time Hermite optical flow approach. This organic and nature-inspired control strategy was compared with a conventional controller and validated in an educational robot platform, providing excellent results when navigating in dynamic environments with a single-constrained perception device

Development of media production processes for CAR-T therapies

Many of the standard cell culturing unit operations utilized by early stage CAR-T manufacturing processes have been derived from benchtop scale academic processes and require further development to become commercially viable. Critical unit operations, such as isolation, activation, transduction, and expansion are often the focus of next generation or automation technologies. Development of ancillary processes such as medium production, however, should not be overlooked and can take advantage of economies of scale and technologies that have been proven in other pharmaceutical industries like biologics. Special consideration should be taken when developing these medium scale-up processes since cell therapies are complex and can be highly sensitive to medium composition changes. In addition, significant changes may be needed to update medium production processes from a process suited for an academic setting to one suited for a commercialized product. This poster discusses Celgene’s approach for developing a commercially sustainable media preparation process by applying available filtration and bulk solution preparation technologies and the unique challenges associated with applying these technologies to CAR-T therapies