Factors affecting product association as a mechanism of host-cell protein persistence in bioprocessing.

Product association of host-cell proteins (HCPs) to monoclonal antibodies (mAbs) is widely regarded as a mechanism that can enable HCP persistence through multiple purification steps and even into the final drug substance. Discussion of this mechanism often implies that the existence or extent of persistence is directly related to the strength of binding but actual measurements of the binding affinity of such interactions remain sparse. Two separate avenues of investigation of HCP-mAb binding are reported here. One is the measurement of the affinity of binding of individual, commonly persistent Chinese hamster ovary (CHO) HCPs to each of a set of mAbs, and the other uses quantitative proteomic measurements to assess binding of HCPs in a null CHO harvested cell culture fluid (HCCF) to mAbs produced in the same cell line. The individual HCP measurements show that the binding affinities of individual HCPs to different mAbs can vary appreciably but are rarely very high, with only weak pH dependence. The measurements on the null HCCF allow estimation of individual HCP-mAb affinities; these are typically weaker than those seen in affinity measurements on isolated HCPs. Instead, the extent of binding appears correlated with the initial abundance of individual HCPs in the HCCF and the forms of the HCPs in the solution, i.e., whether HCPs are present as free molecules or as parts of large aggregates. Separate protein A chromatography experiments performed by feeding different fractions of a mAb-containing HCCF obtained by size-exclusion chromatography (SEC) showed clear differences in the number and identity of HCPs found in the protein A eluate. These results indicate a significant role for HCP-mAb association in determining HCP persistence through protein A chromatography, presumably through binding of HCP-mAb complexes to the resin. Overall, the results illustrate the importance of considering more fully the biophysical context of HCP-product association in assessing the factors that may affect the phenomenon and determine its implications. Knowledge of the abundances and the forms of individual or aggregated HCPs in HCCF are particularly significant, emphasizing the integration of upstream and downstream bioprocessing and the importance of understanding the collective properties of HCPs in addition to just the biophysical properties of individual HCPs.

Identification and characterization of CHO host-cell proteins in monoclonal antibody bioprocessing.

Host-cell proteins (HCPs) are the foremost class of process-related impurities to be controlled and removed in downstream processing steps in monoclonal antibody (mAb) manufacturing. However, some HCPs may evade clearance in multiple purification steps and reach the final drug product, potentially threatening drug stability and patient safety. This study extends prior work on HCP characterization and persistence in mAb process streams by using mass spectrometry (MS)-based methods to track HCPs through downstream processing steps for seven mAbs that were generated by five different cell lines. The results show considerable variability in HCP identities in the processing steps but extensive commonality in the identities and quantities of the most abundant HCPs in the harvests for different processes. Analysis of HCP abundance in the harvests shows a likely relationship between abundance and the reproducibility of quantification measurements and suggests that some groups of HCPs may hinder the characterization. Quantitative monitoring of HCPs persisting through purification steps coupled with the findings from the harvest analysis suggest that multiple factors, including HCP abundance and mAb-HCP interactions, can contribute to the persistence of individual HCPs and the identification of groups of common, persistent HCPs in mAb manufacturing.

Comparison of vector elements and process conditions in transient and stable suspension HEK293 platforms using SARS-CoV-2 receptor binding domain as a model protein.

BACKGROUND: Mammalian cell lines are frequently used as protein expression hosts because of their ability to correctly fold and assemble complex proteins, produce them at high titers, and confer post-translational modifications (PTMs) critical to proper function. Increasing demand for proteins with human-like PTMs, particularly viral proteins and vectors, have made human embryonic kidney 293 (HEK293) cells an increasingly popular host. The need to engineer more productive HEK293 platforms and the ongoing nature of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic presented an opportunity to study strategies to improve viral protein expression in transient and stable HEK293 platforms. RESULTS: Initial process development was done at 24 deep well plate (DWP) -scale to screen transient processes and stable clonal cell lines for recombinant SARS-CoV-2 receptor binding domain (rRBD) titer. Nine DNA vectors that drove rRBD production under different promoters and optionally contained Epstein-Barr virus (EBV) elements to promote episomal expression were screened for transient rRBD production at 37 °C or 32 °C. Use of the cytomegalovirus (CMV) promoter to drive expression at 32 °C led to the highest transient protein titers, but inclusion of episomal expression elements did not augment titer. In parallel, four clonal cell lines with titers higher than that of the selected stable pool were identified in a batch screen. Flask-scale transient transfection and stable fed-batch processes were then established that produced rRBD up to 100 mg/L and 140 mg/L, respectively. While a bio-layer interferometry (BLI) assay was crucial for efficiently screening DWP batch titers, an enzyme-linked immunosorbent assay (ELISA) was used to compare titers from the flask-scale batches due to varying matrix effects from different cell culture media compositions. CONCLUSION: Comparing yields from the flask-scale batches revealed that stable fed-batch cultures produced up to 2.1x more rRBD than transient processes. The stable cell lines developed in this work are the first reported clonal, HEK293-derived rRBD producers and have titers up to 140 mg/L. As stable production platforms are more economically favorable for long-term protein production at large scales, investigation of strategies to increase the efficiency of high-titer stable cell line generation in Expi293F or other HEK293 hosts is warranted.

High-efficiency and multilocus targeted integration in CHO cells using CRISPR-mediated donor nicking and DNA repair inhibitors.

Efforts to leverage clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) for targeted genomic modifications in mammalian cells are limited by low efficiencies and heterogeneous outcomes. To aid method optimization, we developed an all-in-one reporter system, including a novel superfolder orange fluorescent protein (sfOrange), to simultaneously quantify gene disruption, site-specific integration (SSI), and random integration (RI). SSI strategies that utilize different donor plasmid formats and Cas9 nuclease variants were evaluated for targeting accuracy and efficiency in Chinese hamster ovary cells. Double-cut and double-nick donor formats significantly improved targeting accuracy by 2.3-8.3-fold and 19-22-fold, respectively, compared to standard circular donors. Notably, Cas9-mediated donor linearization was associated with increased RI events, whereas donor nicking minimized RI without sacrificing SSI efficiency and avoided low-fidelity outcomes. A screen of 10 molecules that modulate the major mammalian DNA repair pathways identified two inhibitors that further enhance targeting accuracy and efficiency to achieve SSI in 25% of transfected cells without selection. The optimized methods integrated transgene expression cassettes with 96% efficiency at a single locus and with 53%-55% efficiency at two loci simultaneously in selected clones. The CRISPR-based tools and methods developed here could inform the use of CRISPR/Cas9 in mammalian cell lines, accelerate mammalian cell line engineering, and support advanced recombinant protein production applications.

A compendium of stable hotspots in the CHO genome.

The use of targeted integration for industrial CHO cell line development currently requires significant upfront effort to identify genomic loci capable of supporting multigram per liter therapeutic protein production from a limited number of transgene copies. To address this barrier to widespread adoption, we characterized transgene expression from thousands of stable hotspots in the CHO genome using the Thousands of Reporters Integrated in Parallel high-throughput screening method. This genome-scale data set was used to define a limited set of epigenetic properties of hotspot regions with sizes on the order of 10 kb. Cell lines with landing pad integrations at eight retargeted hotspot candidates consistently exhibited higher transgene mRNA expression than a commercially viable hotspot in equivalent culture conditions. Initial benchmarking of NISTmAb and trastuzumab productivity from one of these hotspots yielded mAb productivities of approximately 0.7-2 g/L (qP range: 2.9-8.2 pg/cell/day) in small-scale fed-batches. These findings indicate the list of hotspot candidates identified here will be a valuable resource for targeted integration platform development within the CHO community.

Driving adoption of new technologies in biopharmaceutical manufacturing.

The challenge of introducing new technologies into established industries is not a problem unique to the biopharmaceutical industry. However, it may be critical to the long-term competitiveness of individual manufacturers and, more importantly, the ability to deliver therapies to patients. This is especially true for new treatment modalities including cell and gene therapies. We review several barriers to technology adoption which have been identified in various public forums including business, regulatory, technology, and people-driven concerns. We also summarize suitable enablers addressing one or more of these barriers along with suggestions for developing synergies or connections between innovation in product discovery and manufacturing or across the supplier, discovery, manufacturing, and regulatory arms of the holistic innovation engine.

Design space determination to optimize DNA complexation and full capsid formation in transient rAAV manufacturing.

Recombinant adeno-associated virus (rAAV) vectors are a promising platform for in vivo gene therapies. However, cost-effective, well-characterized processes necessary to manufacture rAAV therapeutics are challenging to develop without an understanding of how process parameters (PPs) affect rAAV product quality attributes (PQAs). In this work, a central composite orthogonal experimental design was employed to examine the influence of four PPs for transient transfection complex formation (polyethylenimine:DNA [PEI:DNA] ratio, total DNA/cell, cocktail volume, and incubation time) on three rAAV PQAs related to capsid content (vector genome titer, vector genome:capsid particle ratio, and two-dimensional vector genome titer ratio). A regression model was established for each PQA using partial least squares, and a design space (DS) was defined in which Monte Carlo simulations predicted < 1% probability of failure (POF) to meet predetermined PQA specifications. Of the three PQAs, viral genome titer was most strongly correlated with changes in complexation PPs. The DS and acceptable PP ranges were largest when incubation time and cocktail volume were kept at mid-high setpoints, and PEI:DNA ratio and total DNA/cell were at low-mid setpoints. Verification experiments confirmed model predictive capability, and this work establishes a framework for studying other rAAV PPs and their relationship to PQAs.

Characterization and implications of host-cell protein aggregates in biopharmaceutical processing.

In the production of biopharmaceuticals such as monoclonal antibodies (mAbs) and vaccines, the residual amounts of host-cell proteins (HCPs) are among the critical quality attributes. In addition to overall HCP levels, individual HCPs may elude purification, potentially causing issues in product stability or patient safety. Such HCP persistence has been attributed mainly to biophysical interactions between individual HCPs and the product, resin media, or residual chromatin particles. Based on measurements on process streams from seven mAb processes, we have found that HCPs in aggregates, not necessarily chromatin-derived, may play a significant role in the persistence of many HCPs. Such aggregates may also hinder accurate detection of HCPs using existing proteomics methods. The findings also highlight that certain HCPs may be difficult to remove because of their functional complementarity to the product; specifically, chaperones and other proteins involved in the unfolded protein response (UPR) are disproportionately present in the aggregates. The methods and findings described here expand our understanding of the origins and potential behavior of HCPs in cell-based biopharmaceutical processes and may be instrumental in improving existing techniques for HCP detection and clearance.

Generation of reference cell lines, media, and a process platform for CHO cell biomanufacturing.

Due to the favorable attributes of Chinese hamster ovary (CHO) cells for therapeutic proteins and antibodies biomanufacturing, companies generate proprietary cells with desirable phenotypes. One key attribute is the ability to stably express multi-gram per liter titers in chemically defined media. Cell, media, and feed diversity has limited community efforts to translate knowledge. Moreover, academic, and nonprofit researchers generally cannot study "industrially relevant" CHO cells due to limited public availability, and the time and knowledge required to generate such cells. To address these issues, a university-industrial consortium (Advanced Mammalian Biomanufacturing Innovation Center, AMBIC) has acquired two CHO "reference cell lines" from different lineages that express monoclonal antibodies. These reference cell lines have relevant production titers, key performance outcomes confirmed by multiple laboratories, and a detailed technology transfer protocol. In commercial media, titers over 2 g/L are reached. Fed-batch cultivation data from shake flask and scaled-down bioreactors is presented. Using productivity as the primary attribute, two academic sites aligned with tight reproducibility at each site. Further, a chemically defined media formulation was developed and evaluated in parallel to the commercial media. The goal of this work is to provide a universal, industrially relevant CHO culture platform to accelerate biomanufacturing innovation.

Comprehensive assessment of host cell protein expression after extended culture and bioreactor production of CHO cell lines.

The biomanufacturing industry is advancing toward continuous processes that will involve longer culture durations and older cell ages. These upstream trends may bring unforeseen challenges for downstream purification due to fluctuations in host cell protein (HCP) levels. To understand the extent of HCP expression instability exhibited by Chinese hamster ovary (CHO) cells over these time scales, an industry-wide consortium collaborated to develop a study to characterize age-dependent changes in HCP levels across 30, 60, and 90 cell doublings, representing a period of approximately 60 days. A monoclonal antibody (mAb)-producing cell line with bulk productivity up to 3 g/L in a bioreactor was aged in parallel with its parental CHO-K1 host. Subsequently, both cell types at each age were cultivated in an automated bioreactor system to generate harvested cell culture fluid (HCCF) for HCP analysis. More than 1500 HCPs were quantified using complementary proteomic techniques, two-dimensional electrophoresis (2DE) and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). While up to 13% of proteins showed variable expression with age, more changes were observed when comparing between the two cell lines with up to 47% of HCPs differentially expressed. A small subset (50 HCPs) with age-dependent expression were previously reported to be problematic as high-risk and/or difficult-to-remove impurities; however, the vast majority of these were downregulated with age. Our findings suggest that HCP expression changes over this time scale may not be as dramatic and pose as great of a challenge to downstream processing as originally expected but that monitoring of variably expressed problematic HCPs remains critical.

Biomanufacturing readiness levels [BRL]-A shared vocabulary for biopharmaceutical technology development and commercialization.

The Manufacturing Readiness Levels (MRLs) developed by the Department of Defense are well-established tools for describing the maturity of new technologies resulting from government-sponsored Research and Development programs, from the concept phase to commercial deployment. While MRLs are generally applicable to a wide range of industries and technologies, there is significant value in offering an industry-specific view on how the basic principles may be applied to biomanufacturing. This paper describes Biomanufacturing Readiness Levels (BRLs) developed by the National Institute for Innovation in Manufacturing Biopharmaceuticals (NIIMBL), a public/private partnership that is part of the Manufacturing USA network. NIIMBL brings together private, federal, nonprofit, and academic stakeholders to accelerate the deployment of innovative technologies for biopharmaceutical production and to educate and train a world-leading biomanufacturing workforce. We anticipate that these BRLs will lay the groundwork for a shared vocabulary for assessment of technology maturity and readiness for commercial biomanufacturing that effectively meets the needs of this critical, specialized, and highly regulated industry.

Restoration of DNA repair mitigates genome instability and increases productivity of Chinese hamster ovary cells.

Chinese hamster ovary (CHO) cells are the primary host for manufacturing of therapeutic proteins. However, productivity loss is a major problem and is associated with genome instability, as chromosomal aberrations reduce transgene copy number and decrease protein expression. We analyzed whole-genome sequencing data from 11 CHO cell lines and found deleterious single-nucleotide variants in DNA repair genes. Comparison with primary Chinese hamster cells confirmed DNA repair to be compromised in CHO. Correction of key DNA repair genes by single-nucleotide variant reversal or expression of intact complementary DNAs successfully improved DNA repair and mitigated karyotypic instability. Moreover, overexpression of intact copies of LIG4 and XRCC6 in a CHO cell line expressing secreted alkaline phosphatase mitigated transgene copy loss and improved protein titer retention. These results show that correction of DNA repair genes yields improvements in genome stability in CHO, and provide new opportunities for cell line development for sustainable protein expression.

Bioindustrial manufacturing readiness levels (BioMRLs) as a shared framework for measuring and communicating the maturity of bioproduct manufacturing processes.

Readiness level (RL) frameworks such as technology readiness levels and manufacturing readiness levels describe the status of a technology/manufacturing process on its journey from initial conception to commercial deployment. More importantly, they provide a roadmap to guide technology development and scale-up from a ''totality of system'' approach. Commercialization risks associated with too narrowly focused R&D efforts are mitigated. RLs are defined abstractly so that they can apply to diverse industries and technology sectors. However, differences between technology sectors make necessary the definition of sector specific RL frameworks. Here, we describe bioindustrial manufacturing readiness levels (BioMRLs), a classification system specific to bioindustrial manufacturing. BioMRLs will give program managers, investors, scientists, and engineers a shared vocabulary for prioritizing goals and assessing risks in the development and commercialization of a bioindustrial manufacturing process.

EvalDNA: a machine learning-based tool for the comprehensive evaluation of mammalian genome assembly quality.

BACKGROUND: To select the most complete, continuous, and accurate assembly for an organism of interest, comprehensive quality assessment of assemblies is necessary. We present a novel tool, called Evaluation of De Novo Assemblies (EvalDNA), which uses supervised machine learning for the quality scoring of genome assemblies and does not require an existing reference genome for accuracy assessment. RESULTS: EvalDNA calculates a list of quality metrics from an assembled sequence and applies a model created from supervised machine learning methods to integrate various metrics into a comprehensive quality score. A well-tested, accurate model for scoring mammalian genome sequences is provided as part of EvalDNA. This random forest regression model evaluates an assembled sequence based on continuity, completeness, and accuracy, and was able to explain 86% of the variation in reference-based quality scores within the testing data. EvalDNA was applied to human chromosome 14 assemblies from the GAGE study to rank genome assemblers and to compare EvalDNA to two other quality evaluation tools. In addition, EvalDNA was used to evaluate several genome assemblies of the Chinese hamster genome to help establish a better reference genome for the biopharmaceutical manufacturing community. EvalDNA was also used to assess more recent human assemblies from the QUAST-LG study completed in 2018, and its ability to score bacterial genomes was examined through application on bacterial assemblies from the GAGE-B study. CONCLUSIONS: EvalDNA enables scientists to easily identify the best available genome assembly for their organism of interest without requiring a reference assembly. EvalDNA sets itself apart from other quality assessment tools by producing a quality score that enables direct comparison among assemblies from different species.

Systematic identification of safe harbor regions in the CHO genome through a comprehensive epigenome analysis.

The Chinese hamster ovary (CHO) cell lines that are used to produce commercial quantities of therapeutic proteins commonly exhibit a decrease in productivity over time in culture, a phenomenon termed production instability. Random integration of the transgenes encoding the protein of interest into locations in the CHO genome that are vulnerable to genetic and epigenetic instability often causes production instability through copy number loss and silencing of expression. Several recent publications have shown that these cell line development challenges can be overcome by using site-specific integration (SSI) technology to insert the transgenes at genomic loci, often called "hotspots," that are transcriptionally permissive and have enhanced stability relative to the rest of the genome. However, extensive characterization of the CHO epigenome is needed to identify hotspots that maintain their desirable epigenetic properties in an industrial bioprocess environment and maximize transcription from a single integrated transgene copy. To this end, the epigenomes and transcriptomes of two distantly related cell lines, an industrially relevant monoclonal antibody-producing cell line and its parental CHO-K1 host, were characterized using high throughput chromosome conformation capture and RNAseq to analyze changes in the epigenome that occur during cell line development and associated changes in system-wide gene expression. In total, 10.9% of the CHO genome contained transcriptionally permissive three-dimensional chromatin structures with enhanced genetic and epigenetic stability relative to the rest of the genome. These safe harbor regions also showed good agreement with published CHO epigenome data, demonstrating that this method was suitable for finding genomic regions with epigenetic markers of active and stable gene expression. These regions significantly reduce the genomic search space when looking for CHO hotspots with widespread applicability and can guide future studies with the goal of maximizing the potential of SSI technology in industrial production CHO cell lines.

Chromosome-scale scaffolds for the Chinese hamster reference genome assembly to facilitate the study of the CHO epigenome.

The Chinese hamster genome serves as a reference genome for the study of Chinese hamster ovary (CHO) cells, the preferred host system for biopharmaceutical production. Recent re-sequencing of the Chinese hamster genome resulted in the RefSeq PICR meta-assembly, a set of highly accurate scaffolds that filled over 95% of the gaps in previous assembly versions. However, these scaffolds did not reach chromosome-scale due to the absence of long-range scaffolding information during the meta-assembly process. Here, long-range scaffolding of the PICR Chinese hamster genome assembly was performed using high-throughput chromosome conformation capture (Hi-C). This process resulted in a new "PICRH" genome, where 97% of the genome is contained in 11 mega-scaffolds corresponding to the Chinese hamster chromosomes (2n = 22) and the total number of scaffolds is reduced by three-fold from 1,830 scaffolds in PICR to 647 in PICRH. Continuity was improved while preserving accuracy, leading to quality scores higher than recent builds of mouse chromosomes and comparable to human chromosomes. The PICRH genome assembly will be an indispensable tool for designing advanced genetic engineering strategies in CHO cells and enabling systematic examination of genomic and epigenomic instability through comparative analysis of CHO cell lines on a common set of chromosomal coordinates.

Adsorptive-Mediated Endocytosis of Sulfo-Cy5-Labeled IgG Causes Aberrant IgG Processing by Brain Endothelial-Like Cells.

Brain endothelial cells (BECs) hinder macromolecules from reaching brain parenchyma, necessitating the evaluation and engineering of therapeutic immunoglobulin γ (IgG) for improved brain delivery. Emerging fluorescent-based approaches to assess IgG brain exposure can expedite and complement current methods; however, alterations in IgG pharmacokinetics following fluorophore conjugation, which remain unexplained, indicate that conjugation may confound analysis of native IgG processing. Here, changes in transcytosis and intracellular processing of IgG conjugates (with sulfonated cyanine 5) were examined using human induced pluripotent stem cell-derived BECs (iBECs). Above a critical degree of labeling, transcytosis rates increased significantly but could be attenuated by nonspecific protein competition. Concurrent increases in intracellular accumulation, which was not attributable to disrupted binding by the neonatal Fc receptor (FcRn), are indicative of indirect reduction of FcRn-mediated recycling that agrees with reported aberrations in the pharmacokinetics of certain unconjugated IgGs. Overall, these findings support the notion that certain fluorophore-IgG conjugates can engage in adsorptive interactions with cell surface moieties, reminiscent of phenomena exhibited by cationized IgG, and provide in vitro criteria to identify changes in IgG processing following fluorophore conjugation.

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.