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Strong, stiff & auxetic - Lessons learned from a fascinating biological material
Get PDFMaterials that display a negative Poisson’s ratio, a property also referred to as auxeticity, have been found in nature and created in engineering through various structural mechanisms. However, these examples, such as cow skin or kirigami structures, are typically low-stiffness designs, since the structural unit rotation or folding usually requires open space for accommodation. Thus, uniting auxeticity with high strength and high stiffness has remained challenging. In this work, we focus on the properties of the limpet teeth, as this material excels in rather harsh loading situations. We combine in-situ nanomechanical testing in SEM and TEM with high resolution structural investigations and microstructure-based modeling to show that the leading part of the limpet teeth successfully achieves this rare combination of properties
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Environmental reliability and crack propagation resistance of 3d-printed ALD-coated nano-ceramics
Get PDF3D-printed micro- and nano-architected ceramic metamaterials currently emerge as a class of lightweight materials with exceptional strength and stiffness. However, their application is hampered by the lack of knowledge of their mechanical reliability. Recently, the sensitivity of nano-ceramics’ crack propagation resistance to environmental conditions, triggered by the unavoidable presence of surface flaws introduced by the TPP-DLW 3D printing and pyrolization post-processing, has been evidenced [1], with a reduction of 20% in the average fracture toughness value reported at high relative humidity levels of testing ( 60%) from the generally performed low-humidity-based testing.
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Rapid product characterization for release using membrane microscopy
Get PDFCell, protein, and viral aggregates are critical quality attributes for all biological products. Subvisible biotherapeutic product aggregates indicate low product stability and low shelf life. In addition, these attributes are a crucial indicator of potential immunogenicity for a given biological drug. The FDA suggests that “strategies to minimize aggregate formation should be developed as early as feasible in product development.” Cell therapies present a unique challenge in that cells themselves are subvisible in nature, and distinguishing cells vs. large cellular aggregates and other product impurities remains a challenge, until now. Aura™ is the first system specifically designed to count, characterize, and ID particles in a rapid and low-volume assay by combining membrane microscopy with labeled fluorescence.
Here, we demonstrate how to the Aura quickly and accurately finds trace amounts of subvisible and visible particle contaminants in cell and gene therapy materials. Some examples include residual Dynabeads in CAR-T cell products, identifying the presence of extraneous fibers, and lentiviral aggregates. Aura enables the ability to characterize, size and identify every particle in across every cell and gene therapy experiment
Proof-of-concept of a novel scalable magnetic bead-based cell separation technology
Get PDFAdvanced Therapy Medicinal Products (ATMPs) are gaining great interest for the treatment of severe, currently consider incurable, diseases. Therapies based on stem cells have an enormous potential in applications such as cardiac cells and neurons to name a few. However, the production of these cell systems is expensive, complex and lack, nowadays, scalability both for their cultivation and the purification. The lack of scalability is a major bottleneck to bring these therapies to patient at commercial scale.
Magnetic beads are well-established for sorting of cells, e.g. magnetic activated cell sorting. However, today´s systems size is still limited in terms of scale-up potential. We have developed a new scalable separation process based on the magnetic bead MAG for the isolation of receptor positive cell subpopulations. We have previously published that our new magnetic bead system MAG is extremely gentle towards cells1 and can easily be scaled up at pilot-scale for the separation of monoclonal antibody from a cell suspension2. In the present study, this magnetic bead system has been further developed for cell separation. In a model system with a mixture of hMSC and HER2+ SK BR3 cells (20:80), a proof-of-concept was demonstrated showing exceptional elimination of the HER2+ cells. Different ligand densities were evaluated, showing that the largest cell removals were achieved with the lowest ligand densities. Furthermore, in a study of mechanical and chemical stress conditions, the MAG separation system showed robustness of sorting performances. From our previous knowledge about the scalability of the MAG magnetic beads separation, this provides promising potential for the production of therapeutic stem cells at larger scale.
1. Brechmann, N. A.; Schwarz, H.; Eriksson, P.-O.; Eriksson, K.; Shokri, A.; Chotteau, V., Antibody capture process based on magnetic beads from very high cell density suspension. Biotechnology and Bioengineering 2021, n/a, (n/a).
2. Brechmann, N. A.; Eriksson, P.-O.; Eriksson, K.; Oscarsson, S.; Buijs, J.; Shokri, A.; Hjälm, G.; Chotteau, V., Pilot-scale process for magnetic bead purification of antibodies directly from non-clarified CHO cell culture. Biotechnology Progress 2019, 35, (3), e2775
Downstream Improvement for Recombinant Adeno-Associated Viruses (rAAV) Produced in iCELLis Nano 4 m2 Adherent Bioreactor
Get PDFIn the clarification of recombinant adeno-associated virus cell culture (rAAV), unwanted cellular material is separated from the vector to increase its purity and enable further downstream processing. In industrial settings, primary clarification with depth filters is used to remove larger particles, such as cells and cellular debris originating from cell lysis and benzonase treatment. To reduce the challenge on the downstream process a clarification step that can eliminate these contaminants with minimal rAAV loss is highly desirable for robust GMP manufacture. We are working towards improving the clarification and downstream steps for rAAV5, 9 and other rAAV subtypes. We produced rAAV on the iCELLis Nano 4 m2 adherent bioreactor with 8 L media. At the point of harvest, the cells were lysed and were treated with benzonase. ‘The upstream feed was assessed for turbidity and subsequently processed and concentrated by depth filtration and Tangential Flow Filtration (TFF) and the reduction in turbidity determined
Enhancing rAAV production by HEK293 cells via metabolic profiling
Get PDFViral vector manufacturing is expensive and time-consuming. Demand for rAAV-based vectors has risen
massively in the past decade and continues to rise thanks to urgent healthcare supply demand. The industry is,
however, currently missing a cost-effective and robust manufacturing strategy. One of the major downsides of
rAAV production is the high percentage of “empty” vector particles being produced and harvested. In addition to
complicating downstream purification processes, this characteristic limits the efficiency of rAAV manufacture
and presents uncertainties for scale-up.
Efficiency of the manufacturing process is largely dependent on the productivity of the production cell line. Much
emphasis has been put into understanding the effects of recombinant protein production on mammalian cell
lines (e.g., CHO, HeLa, HEK293) but relatively little is known about the effects of viral vector production on cell
biology and behaviour. Over the years, many clones have been derived, isolated and engineered from HEK293
to induce improvements in productivity and efficiency. However, the high cost of production and licensing, the
expression of potentially undesired elements (e.g., T-antigen) and regulatory approval processes for next
generation cell lines, hinders their use in clinical manufacturing. Increased understanding of HEK293 in relation
to existing processes and process control offers realistic opportunity to enhance the efficiency of rAAV
manufacturing.
Our aim is to identify and understand the critical parameters that contribute to setting the productivity in HEK293
cells (in terms of final yield and abundance of full capsids), ranging from the metabolic requirements prior to and
during viral vector production, to cell culture parameter optimisation to maintain the cells in an optimal state of
health.
We tested several commercially available media for rAAV9 production and selected the candidate that provided
the best yield and quality of viral vector. With this medium as our baseline, we investigated the metabolism
during a period of culture via extracellular metabolic profiling of control and rAAV producing cells. The analysis
revealed the rapid use of several amino acids over the first 24 hr post-inoculation and the subsequent
generation of metabolites indicative of metabolic profiles associated with cell growth. rAAV9 producing cells
show lower rates of amino acid and glucose consumption than control cells but the profile of metabolism was
not significantly changed as a result of transfection/production of rAAV9. These data were used to design
medium supplements and the effect of supplement addition on cell proliferation, viability and rAAV
production/quality was assessed. Specific combinations of amino acids generated an increased cell density (up
to 9.3x106 cells/mL at 5 days post-inoculation compared to 4.4x106 cells/mL for cells in non-supplemented
medium). This was associated with retention of improved viability in the presence of the supplement. In addition,
the metabolic profiling we undertook indicated the build-up of potentially toxic/growth inhibitory metabolites
during the period of stock cell preparations prior to setting up transfections. In various dilution experiments we
were able to optimise the pre-treatment, cell density and dilution protocol to generate predictable and
reproducible efficiencies of transfection, cell growth and rAAV production.
Overall, our data contributes metabolic insights to process conditions that generate HEK293 cells of appropriate
health and defined parameters to robust and enhanced production of rAAV, providing work schemes that are
also appropriate to the manufacture of further types of viral vectors
The role of NIIMBL to advance manufacturing for Cell and Gene Therapy treatments
Get PDFThe National Institute for Innovation in Manufacturing Biopharmaceuticals (NIIMBL) is a public-private partnership whose mission is to accelerate biopharmaceutical innovation, support the development of standards that enable more efficient and rapid manufacturing capabilities, and educate and train a world-leading biopharmaceutical manufacturing workforce, fundamentally advancing U.S. competitiveness in this industry.
Funding of $70 million has been committed by NIST for the next 5 years of NIIMBL and this is expected to be more than matched by participants. A key focus will be advancing manufacturing of Gene Therapy and Cell Therapy based products. Discussion is invited regarding the best role for this public-private partnership
Development of a disruptive mass photometry technology for AAV empty full quantification
Get PDFProduct-related impurities (empty capsids) are a safety risk and the ability to quantify the removal of empty AAV particles is a critical requirement for product release. Traditional methods such as PCR and protein quantification are labour intensive and compound the inherent errors of the individual assays. The collaborative project between Refeyn and CGT Catapult included use of commercially available mass photometry instrument as well as developments aiming specifically at weighing large molecules, including AAV. We generated accurate Empty and Full particle quantification for different AAV serotypes and at different purification levels. Empty:Full ratio is generated in 5 minutes and requires minimal sample volume and preparation. Our current data correlates with the gold-standard cryoTEM and AUC analytical characterisation, circumventing the requirement of large capital expense and skilled operators for operation and data analysis
