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Process and analytical development challenges for the incorporation of gene edits into T cell therapies
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Extra cellular vesicles separation and biophysical characterization
Get PDFCells bud off up to 10-fold of their biomass in form of extracellular vesicles. The term ‘extracellular vesicles’ refers to a heterogeneous population of vesicular bodies of cellular origin that derive either from the endosomal compartment (exosomes) or as a result of shedding from the plasma membrane (microvesicles, oncosomes and apoptotic bodies). Extracellular vesicles carry a variety of cargo, including RNA, proteins, lipids and DNA, which can be taken up by other cells, both in the direct vicinity of the source cell and at distant sites in the body via biofluids, and stimulate a variety of phenotypic responses. These functions of extracellular vesicles are not necessary when single cells are produced in a bioreactor for the plain purpose of biomass generation. The fact that cells in in-vitro culture release that many extracellular vesicles is completely neglected by research in the life science and biochemical engineering community. In addition, a lot of chromatin is present in cell culture supernatant. Due to the presence of chromatin the particle count is always overestimated due to the similar size between chromatin and extra cellular vesicles.
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Enhancements for a more reliable single-use pH sensor
Get PDFThe adoption of single-use bioreactors is increasing in the pharmaceutical process industry. Advantages of single-use technology have resulted in an increasing demand for improved pH sensors in single-use format. Sensors that are fully integrated into a single-use bioreactor bag facilitate the workflow for sensor handling and operation at end-users’ sites, and mitigate the risk of breaching the sterile integrity of single-use bioreactors.
Until now, mainly single-use pH sensor spots or patches based on optical measurement technology have been integrated in single-use process devices. But in recent years, the market for single-use pH sensors has been characterized by an increasing availability of electrochemical glass pH sensors – in principle, a single-use equivalent to the well-established and characterized reusable in-line pH glass sensors that are employed in the industry for process monitoring and control purposes. However, applying this technology for a single-use sensor necessitates meeting the more stringent requirements for single-use process devices, such as a minimum of two years dry storage capacity (shelf life), availability of post-gamma extractables study data, reliable measurement performance after gamma irradiation and dry storage, and maintaining single-use bioreactors\u27 integrity. The last point is very important, because the integration of a glass pH sensor always leads to the release of certain materials of the sensor components into the process media. For example, electrolyte from the sensor’s reference electrode, sensor storage solution or electrode passivation coating material. Furthermore, the risk of glass breakage must be mitigated during single-use bioreactor bag transport, storage and operation. This means that the influence of these factors must be understood and considered in the development phase of single-use pH sensors.
In our contribution, we will discuss and present a single-use pH sensor that was developed to meet these stringent requirements. We will provide:
- How the sensor’s form factor leads to its safe integration into single-use bioreactors.
- How the passivation of the sensor’s reference electrode enables a dry storage period of 30 months.
- Post-gamma measurement characterization data after 30 months\u27 dry storage.
- Material compliance data and results of a post-gamma BPOG extractables study
Bioprocess characterization at the micro-scale: Optical sensor integration in a novel capillary-wave micro-bioreactor
Get PDFDue to the high demand of new biopharmaceuticals and bioproducts, the development of new cultivation platforms for high-throughput screenings, cell-based assays and bioprocess development is of high interest. Therefore, micro-bioreactors (MBRs) are a promising alternative to conventional cultivation platforms like shake flasks due to their minimal volume, sensor integration and high ability for automatization and parallelization. Especially, MBRs with a volume below 10 µL can reduce the amount of needed testing substances for cell-based assays, which is advantageous mostly for testing new biopharmaceuticals with limited availability. However, characterization of a cell culture in the lower micro-liter scale is challenging due to the limited space and the insufficient volume for sampling and offline analysis. Optical sensors are one suitable possibility to close this gap. Therefore, a novel capillary-wave micro-bioreactor (cwMBR) with a working volume of 7 µL and optical sensors for biomass, glucose, oxygen, pH and fluorescence intensity measurement was developed.
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Reduction of water and water-related energy consumption by in-situ media and buffer preparation on demand in continuous integrated
Get PDFFigure 1 – (A) 3D printed single use device for feeding of dry powdered medium or buffer with its (B) top-, (C) front-, (D) and side view.
A strong incentive for continuous integrated biomanufacturing is the improved economics compared to traditional batch wise biomanufacturing. The floor space and the size of unit operations are substantially smaller than in batch. This size reduction allows consequent implementation of single use technology, because single use equipment is not available at large scale biomanufacturing, e.g. reactor volumes above 2000 L or columns bigger than 100 L. However, a point often overlooked is that this transformation only “shrinks” the unit operations itself, while the necessary auxiliaries such as hold tanks, surge vessels and demand of process materials are unchanged or even drastically increased. Hence, the supply chain is facing an increase in demand of process materials and the necessity of handling significantly larger volumes. Media and buffer are always prepared in excess to mitigate the risk of failure for a campaign by running out of it. On a company level, but even more on a global level, this is an enormous waste of resources. We have previously shown that the water consumption is the major contribution to energy demand and CO2 emissions in a bioprocess. This link is described by the so-called metric WARIEN (WAter Related Impact of ENergy), which is also related to the metric PMI (Process Mass Intensity). The reduction and optimization of water consumption bears the highest potential to improve the environmental footprint of biomanufacturing, by reduction of energy consumption and CO2 emission. Here we show a single use device to continuously reconstitute chemically defined media on-demand and buffers directly from solids resulting in the same quality as by conventional batchwise preparation (Figure 1). The long-term operation over a duration of 12 hours demonstrated that such on-demand medium product is robust and precise. This technology with on-demand reconstitution directly from solids will make the repeated preparation of cell culture media and buffers and intermediate hold tanks obsolete which contributes significantly to the reduction of needed floor space. We present an economic and environmental analysis how the on-demand production improves economic and environmental footprint. This is exemplified by manufacturing of antibodies enzymes, hormones and growth factors. Preliminary economic analysis based on Biosolve and SuperPro models that were already built from data coming from the industry the amount of expenses that would be saved yearly on a global scale only by saving medium and buffer is almost 2.8 billion $ if we assume that 20% extra buffer is prepared for each of the bioprocess considered. This number is probably an overestimate but it\u27s just to demonstrate the potential and impact of this technology. The savings by reduction the floorspace are not take into consideration by this economic evaluation. The single use technology and process intensification towards integrated continuous biomanufacturing is a perfect marriage get improved sustainability of biomanufacturing
Building the future with on demand 3D printing
Get PDFThe current shorts in single use (SU) supply chains show how dependent both industry and academia are from only a few vendors worldwide. This is severely hindering fundamental research and process development for the pandemic response. With 3D printing technology we can manufacture SU equipment on demand and on site. In this study we investigated different commercially available low-cost materials and their compatibility for cell culture. We identified poly lactic acid (PLA) as perfect candidate for 3D printed parts for cell culture applications.
The worldwide supply chain issues for SU shaking flasks and reactors gave us the incentive to develop 3D printed counterparts to maintain our HEK293 cell culture. The shake flasks were designed in Autodesk inventor 3D CAD. The materials tested represent the market of different 3D printing technologies and materials, ranging from UV-polymerizing resin printers to thermoplastic printers. We included different manufacturers, plant derived and water washable resins as well as medical Class IIa resins. Whereas resin printed shaking flasks needed washing, curing and sterilization using isopropyl alcohol, the thermoplastic flasks were directly autoclaved. The different materials were tested with HEK293 cells under standard conditions. Cell growth and viability were monitored daily.
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Qualifying X-ray irradiation of single-use systems to address new challenges associated with single-use growth
Get PDFWith single-use technologies playing a pivotal role supporting the rapid development and scale-out of vaccine manufacturing, the experience has highlighted benefits resulting from standardization of SUT requirements and identified key challenges ahead for SUT, including the availability of gamma irradiation capacity. This cobalt-60-dependent gamma irradiation market is led by a relatively small number of market providers, and underpins the entirety of the single-use bioprocess supply chain, regardless of integrator. As the demand for gamma irradiation has quickly outstripped the available industry capacity, significant collaborative industry efforts have been underway to qualify X-ray irradiation as an equivalent alternative to gamma irradiation, such that qualified SUS can be irradiated by either gamma or X-ray, depending on which modality as more readily available at the time of manufacture. In addition to highlighting an industry risk assessment and testing approach, which relies on prior knowledge of the irradiation physics, materials testing, and standardized component testing; data will be shared characterizing the impact of gamma and X-ray irradiation on SUS identifying any similarities and differences in the modalities
Company and assembly redundancy – a case study
Get PDFTakeda’s Massachusetts Biologics Operations (MA Bio Ops) created a task force called Operation Lighthouse to address the unprecedented raw material shortages caused by Covid. Co-lead by supply chain, materials science, and operational excellence partners, Operation Lighthouse created a forum where all solutions – supply options, technical options, and manufacturing process changes – are evaluated, pursued, and implemented by one centralized cross-functional team.
Although the primary goal was to ensure manufacturing continuity, Operation Lighthouse preferentially chose solutions with long-term benefits, such as second sourcing, utilizing additional manufacturing sites from existing suppliers, and accelerating component interchange. This approach required a fundamental shift in thinking to view existing proprietary technologies and materials of construction as an artifact of the original sourcing exercise, rather than an inherent process requirement, thus allowing the team to evaluate multiple options with respect to fitness for use.
As of November 2021, Operation Lighthouse has successfully executed \u3e20 change controls across all material categories, including changes to single-use mixing technologies, bag films, tubing formulations, etc. Our desired end state is to build redundancy into individual assemblies via component interchangeability, to build redundancy into assemblies via multiple suppliers or multiple manufacturing sites within a single supplier, and to partner with our suppliers on these approaches to achieve a long-term state of supply agility that benefits both parties.
Takeda provided all funding for this work
