Summary from Advanced Manufacturing Technology Workshop Held at 6th Accelerating Biopharmaceutical Development Meeting.

A workshop was held at the 6th Accelerating Biopharmaceutical Development meeting in Carlsbad, CA on February 18, 2019. An anonymous survey was sent to all industry participants before the meeting to identify their top technological barriers to achieving a future manufacturing state and a real-time polling tool was used to collect live feedback during the meeting. Senior leaders from across the biopharmaceutical industry discussed the top technology opportunities to enable high-throughput, flexible manufacturing capabilities and business drivers for the industry. New analytical technologies and upstream operations were identified to be opportunities that could have a profound effect on the shift toward flexible manufacturing. Further discussion at the workshop also identified key business drivers, such as speed, simplicity, and flexibility, as well as the global regulatory environment, to be factors that have a major impact on an organization's ability to implement flexible manufacturing. This workshop has identified industry-wide needs and concerns and can inform future industry-wide initiatives and prioritization of resources to achieve more flexible manufacturing.

NIIMBL-Facilitated Active Listening Meeting between Industry and FDA Identifies Common Challenges for Adoption of New Biopharmaceutical Manufacturing Technologies.

The National Institute for Innovation in Manufacturing Biopharmaceuticals (NIIMBL) piloted a forum to encourage an exchange of information between the biopharmaceutical industry and the U.S. Food and Drug Administration (FDA). To facilitate this exchange, NIIMBL conducted a survey of industry representatives around the perceived challenges associated with the adoption of new innovative technologies for biopharmaceutical manufacturing or for continuous improvement and then held an Active Listening session with industry and FDA stakeholders to share common themes. The scope was limited to biotechnology products regulated by the Center for Drug Evaluation and Research (CDER). This manner of exchange has not been tested before and led to meaningful dialog between industry and the Agency and valuable takeaways by all involved. One of the general findings and key points of discussion was around the perceived lack of a business case for adoption of new technology in the manufacture of monoclonal antibodies and therapeutic proteins. Tight timelines were the primary constraints for hesitation around pre-approval implementation and the challenges associated with a global regulatory environment were the primary constraint around post-approval adoption of new technology. Mechanisms that would allow industry and regulatory scientists to develop a shared understanding of new technologies, outside of formal applications, could de-risk adoption of new technologies by the industry. The favorable response to this NIIMBL-facilitated exchange suggests that this format could be useful in establishing a more informal dialog between the FDA and industry on industry-wide challenges.

Cyberbiosecurity for Biopharmaceutical Products.

Cyberbiosecurity is an emerging discipline that addresses the unique vulnerabilities and threats that occur at the intersection of cyberspace and biotechnology. Advances in technology and manufacturing are increasing the relevance of cyberbiosecurity to the biopharmaceutical manufacturing community in the United States. Threats may be associated with the biopharmaceutical product itself or with the digital thread of manufacturing of biopharmaceuticals, including those that relate to supply chain and cyberphysical systems. Here, we offer an initial examination of these cyberbiosecurity threats as they stand today, as well as introductory steps toward paths for mitigation of cyberbiosecurity risk for a safer, more secure future.

Immunoglobulin G transport increases in an in vitro blood-brain barrier model with amyloid-ß and with neuroinflammatory cytokines.

Immunotherapies are a promising strategy for the treatment of neurological diseases such as Alzheimer's disease (AD), however, transport of antibodies to the brain is severely restricted by the blood-brain barrier (BBB). Furthermore, molecular transport at the BBB is altered in disease, which may affect the mechanism and quantity of therapeutic antibody transport. To better understand the transport of immunotherapies at the BBB in disease, an in vitro BBB model derived from human induced pluripotent stem cells (iPSCs) was used to investigate the endocytic uptake route of immunoglobulin G (IgG). In this model, uptake of fluorescently labeled IgGs is a saturable process. Inhibition of clathrin-mediated endocytosis, caveolar endocytosis, and macropinocytosis demonstrated that macropinocytosis is a major transport route for IgGs at the BBB. IgG uptake and transport were increased after the addition of stimuli to mimic AD (Aß1-40 and Aß1-42 ) and neuroinflammation (tumor necrosis factor-α and interleukin-6). Lastly, caveolar endocytosis increased in the AD model, which may be responsible for the increase in IgG uptake in disease. This study presents an iPSC-derived BBB model that responds to disease stimuli with physiologically relevant changes to molecular transport and can be used to understand fundamental questions about transport mechanisms of immunotherapies in health and neurodegenerative disease.

A differentiating neural stem cell-derived astrocytic population mitigates the inflammatory effects of TNF-α and IL-6 in an iPSC-based blood-brain barrier model.

Inflammation can be a risk factor for neurodegenerative diseases such as Alzheimer's disease (AD) and may also contribute to the progression of AD. Here, we sought to understand how inflammation affects the properties of the brain microvascular endothelial cells (BMECs) that compose the blood-brain barrier (BBB), which is impaired in AD. A fully human in vitro BBB model with brain microvascular endothelial cells derived from induced pluripotent stem cells and differentiating neural stem cell (NSC)-derived astrocytic cells was used to investigate the effects of neuroinflammation on barrier function. The cytokines TNF-α and IL-6 directly cause BBB dysfunction measured by a decrease in transendothelial electrical resistance, an increase in sodium fluorescein permeability, and a decrease in cell polarity, providing a link between neuroinflammation and specific aspects of BBB breakdown. An NSC-derived astrocytic cell population was added to the model and secreted cytokines and chemokines were quantified in monoculture and coculture both in the presence and absence of TNF-α and IL-6. Increased concentrations of pro-inflammatory cytokines known to be secreted by astrocytes or endothelial cells such as MCP-1, IL-8, IP-10, MIP-1ß, IL-1 ß, MIG, and RANTES peaked in inflammatory conditions when NSC-astrocytic cells were present. Despite the presence of several pro-inflammatory cytokines, the NSC-derived astrocytic cells mitigated the effects of inflammation measured by a restoration of transendothelial electrical resistance and IgG permeability. These results also suggest a breakdown in transcellular transport that precedes any increase in paracellular permeability in neuroinflammation. This model has the potential to resolve questions about neurodegenerative disease progression and delivery of therapeutics to the brain.

Minimum Transendothelial Electrical Resistance Thresholds for the Study of Small and Large Molecule Drug Transport in a Human in Vitro Blood-Brain Barrier Model.

A human cell-based in vitro model that can accurately predict drug penetration into the brain as well as metrics to assess these in vitro models are valuable for the development of new therapeutics. Here, human induced pluripotent stem cells (hPSCs) are differentiated into a polarized monolayer that express blood-brain barrier (BBB)-specific proteins and have transendothelial electrical resistance (TEER) values greater than 2500 Ω·cm2. By assessing the permeabilities of several known drugs, a benchmarking system to evaluate brain permeability of drugs was established. Furthermore, relationships between TEER and permeability to both small and large molecules were established, demonstrating that different minimum TEER thresholds must be achieved to study the brain transport of these two classes of drugs. This work demonstrates that this hPSC-derived BBB model exhibits an in vivo-like phenotype, and the benchmarks established here are useful for assessing functionality of other in vitro BBB models.

Fabrication of 3D Biomimetic Microfluidic Networks in Hydrogels.

A laser-based hydrogel degradation technique is developed that allows for local control over hydrogel porosity, fabrication of 3D vascular-derived, biomimetic, hydrogel-embedded microfluidic networks, and generation of two intertwining, yet independent, microfluidic networks in a single construct.