Combining descriptive and predictive modeling to systematically design depth filtration-based harvest processes for biologics.

Advances in upstream production of biologics-particularly intensified fed-batch processes beyond 10% cell solids-have severely strained harvest operations, especially depth filtration. Bioreactors containing high amounts of cell debris (more than 40% particles <10 µm in diameter) are increasingly common and drive the need for more robust depth filtration processes, while accelerated timelines emphasize the need for predictive tools to accelerate development. Both needs are constrained by the current limited mechanistic understanding of the harvest filter-feedstream system. Historically, process development relied on screening scale-down depth filter devices and conditions to define throughput before fouling, indicated by increasing differential pressure and/or particle breakthrough (measured via turbidity). This approach is straightforward, but resource-intensive, and its results are inherently limited by the variability of the feedstream. Semi-empirical models have been developed from first principles to describe various mechanisms of filter fouling, that is, pore constriction, pore blocking, and/or surface deposit. Fitting these models to experimental data can assist in identifying the dominant fouling mechanism. Still, this approach sees limited application to guide process development, as it is descriptive, not predictive. To address this gap, we developed a hybrid modeling approach. Leveraging historical bench scale filtration process data, we built a partial least squares regression model to predict particle breakthrough from filter and feedstream attributes, and leveraged the model to demonstrate prediction of filter performance a priori. The fouling models are used to interpret and provide physical meaning to these computational models. This hybrid approach-combining the mechanistic insights of fouling models and the predictive capability of computational models-was used to establish a robust platform strategy for depth filtration of Chinese hamster ovary cell cultures. As new data continues to teach the computational models, in silico tools will become an essential part of harvest process development by enabling prospective experimental design, reducing total experimental load, and accelerating development under strict timelines.

Current state of implementation of in silico tools in the biopharmaceutical industry-Proceedings of the 5th modeling workshop.

The fifth modeling workshop (5MW) was held in June 2023 at Favrholm, Denmark and sponsored by Recovery of Biological Products Conference Series. The goal of the workshop was to assemble modeling practitioners to review and discuss the current state, progress since the last fourth mini modeling workshop (4MMW), gaps and opportunities for development, deployment and maintenance of models in bioprocess applications. Areas of focus were four categories: biophysics and molecular modeling, mechanistic modeling, computational fluid dynamics (CFD) and plant modeling. Highlights of the workshop included significant advancements in biophysical/molecular modeling to novel protein constructs, mechanistic models for filtration and initial forays into modeling of multiphase systems using CFD for a bioreactor and mapped strategically to cell line selection/facility fit. A significant impediment to more fully quantitative and calibrated models for biophysics is the lack of large, anonymized datasets. A potential solution would be the use of specific descriptors in a database that would allow for detailed analyzes without sharing proprietary information. Another gap identified was the lack of a consistent framework for use of models that are included or support a regulatory filing beyond the high-level guidance in ICH Q8-Q11. One perspective is that modeling can be viewed as a component or precursor of machine learning (ML) and artificial intelligence (AI). Another outcome was alignment on a key definition for "mechanistic modeling." Feedback from participants was that there was progression in all of the fields of modeling within scope of the conference. Some areas (e.g., biophysics and molecular modeling) have opportunities for significant research investment to realize full impact. However, the need for ongoing research and development for all model types does not preclude the application to support process development, manufacturing and use in regulatory filings. Analogous to ML and AI, given the current state of the four modeling types, a prospective investment in educating inter-disciplinary subject matter experts (e.g., data science, chromatography) is essential to advancing the modeling community.

Cationic polymer precipitation for enhanced impurity removal in downstream processing.

Precipitation can be used for the removal of impurities early in the downstream purification process of biologics, with the soluble product remaining in the filtrate through microfiltration. The objective of this study was to examine the use of polyallylamine (PAA) precipitation to increase the purity of product via higher host cell protein removal to enhance polysorbate excipient stability to enable a longer shelf life. Experiments were performed using three monoclonal antibodies (mAbs) with different properties of isoelectric point and IgG subclass. High throughput workflows were established to quickly screen precipitation conditions as a function of pH, conductivity and PAA concentrations. Process analytical tools (PATs) were used to evaluate the size distribution of particles and inform the optimal precipitation condition. Minimal pressure increase was observed during depth filtration of the precipitates. The precipitation was scaled up to 20L size and the extensive characterization of precipitated samples after protein A chromatography showed >75% reduction of host cell protein (HCP) concentrations (by ELISA), >90% reduction of number of HCP species (by mass spectrometry), and >99.8% reduction of DNA. The stability of polysorbate containing formulation buffers for all three mAbs in the protein A purified intermediates was improved at least 25% after PAA precipitation. Mass spectrometry was used to obtain additional understanding of the interaction between PAA and HCPs with different properties. Minimal impact on product quality and <5% yield loss after precipitation were observed while the residual PAA was <9 ppm. These results expand the toolbox in downstream purification to solve HCP clearance issues for programs with purification challenges, while also providing important insights into the integration of precipitation-depth filtration and the current platform process for the purification of biologics.

Hemostasis stimulates lymphangiogenesis through release and activation of VEGFC.

Hemostasis associated with tissue injury is followed by wound healing, a complex process by which damaged cellular material is removed and tissue repaired. Angiogenic responses are a central aspect of wound healing, including the growth of new lymphatic vessels by which immune cells, protein, and fluid are transported out of the wound area. The concept that hemostatic responses might be linked to wound healing responses is an old one, but demonstrating such a link in vivo and defining specific molecular mechanisms by which the 2 processes are connected has been difficult. In the present study, we demonstrate that the lymphangiogenic factors vascular endothelial growth factor C (VEGFC) and VEGFD are cleaved by thrombin and plasmin, serine proteases generated during hemostasis and wound healing. Using a new tail-wounding assay to test the relationship between clot formation and lymphangiogenesis in mice, we find that platelets accelerate lymphatic growth after injury in vivo. Genetic studies reveal that platelet enhancement of lymphatic growth after wounding is dependent on the release of VEGFC, but not VEGFD, a finding consistent with high expression of VEGFC in both platelets and avian thrombocytes. Analysis of lymphangiogenesis after full-thickness skin excision, a wound model that is not associated with significant clot formation, also revealed an essential role for VEGFC, but not VEGFD. These studies define a concrete molecular and cellular link between hemostasis and lymphangiogenesis during wound healing and reveal that VEGFC, the dominant lymphangiogenic factor during embryonic development, continues to play a dominant role in lymphatic growth in mature animals.

Noninferiority Designed Cardiovascular Trials in Highest-Impact Journals.

BACKGROUND: Noninferiority trials are increasingly being performed. However, little is known about their methodological quality. We sought to characterize noninferiority cardiovascular trials published in the highest-impact journals, features that may bias results toward noninferiority, features related to reporting of noninferiority trials, and the time trends. METHODS: We identified cardiovascular noninferiority trials published in JAMA, Lancet, or New England Journal of Medicine from 1990 to 2016. Two independent reviewers extracted the data. Data elements included the noninferiority margin and the success of studies in achieving noninferiority. The proportion of trials showing major or minor features that may have affected the noninferiority inference was determined. Major factors included the lack of presenting the results in both intention-to-treat and per-protocol/as-treated cohorts, α>0.05, the new intervention not being compared with the best alternative, not justifying the noninferiority margin, and exclusion or loss of ≥10% of the cohort. Minor factors included suboptimal blinding, allocation concealment, and others. RESULTS: From 2544 screened studies, we identified 111 noninferiority cardiovascular trials. Noninferiority margins varied widely: risk differences of 0.4% to 25%, hazard ratios of 1.05 to 2.85, odds ratios of 1.1 to 2.0, and relative risks of 1.1 to 1.8. Eighty-six trials claimed noninferiority, of which 20 showed superiority, whereas 23 (21.1%) did not show noninferiority, of which 8 also demonstrated inferiority. Only 7 (6.3%) trials were considered low risk for all the major and minor biasing factors. Among common major factors for bias, 41 (37%) did not confirm the findings in both intention-to-treat and per-protocol/as-treated cohorts and 4 (3.6%) reported discrepant results between intention-to-treat and per-protocol analyses. Forty-three (38.7%) did not justify the noninferiority margin. Overall, 27 (24.3%) underenrolled or had >10% exclusions. Sixty trials (54.0%) were open label. Allocation concealment was not maintained or unclear in 11 (9.9%). Publication of noninferiority trials increased over time (P<0.001). Fifty-two (46.8%) were published after 2010 and had a lower risk of methodological or reporting limitations for major (P=0.03) and minor factors (P=0.002). CONCLUSIONS: Noninferiority trials in highest-impact journals commonly conclude noninferiority of the tested intervention, but vary markedly in the selected noninferiority margin, and frequently have limitations that may impact the inference related to noninferiority.

Toward in silico CMC: An industrial collaborative approach to model-based process development.

The Third Modeling Workshop focusing on bioprocess modeling was held in Kenilworth, NJ in May 2019. A summary of these Workshop proceedings is captured in this manuscript. Modeling is an active area of research within the biotechnology community, and there is a critical need to assess the current state and opportunities for continued investment to realize the full potential of models, including resource and time savings. Beyond individual presentations and topics of novel interest, a substantial portion of the Workshop was devoted toward group discussions of current states and future directions in modeling fields. All scales of modeling, from biophysical models at the molecular level and up through large scale facility and plant modeling, were considered in these discussions and are summarized in the manuscript. Model life cycle management from model development to implementation and sustainment are also considered for different stages of clinical development and commercial production. The manuscript provides a comprehensive overview of bioprocess modeling while suggesting an ideal future state with standardized approaches aligned across the industry.

Highland games: A benchmarking exercise in predicting biophysical and drug properties of monoclonal antibodies from amino acid sequences.

Biopharmaceutical product and process development do not yet take advantage of predictive computational modeling to nearly the degree seen in industries based on smaller molecules. To assess and advance progress in this area, spirited coopetition (mutually beneficial collaboration between competitors) was successfully used to motivate industrial scientists to develop, share, and compare data and methods which would normally have remained confidential. The first "Highland Games" competition was held in conjunction with the October 2018 Recovery of Biological Products Conference in Ashville, NC, with the goal of benchmarking and assessment of the ability to predict development-related properties of six antibodies from their amino acid sequences alone. Predictions included purification-influencing properties such as isoelectric point and protein A elution pH, and biophysical properties such as stability and viscosity at very high concentrations. Essential contributions were made by a large variety of individuals, including companies which consented to provide antibody amino acid sequences and test materials, volunteers who undertook the preparation and experimental characterization of these materials, and prediction teams who attempted to predict antibody properties from sequence alone. Best practices were identified and shared, and areas in which the community excels at making predictions were identified, as well as areas presenting opportunities for considerable improvement. Predictions of isoelectric point and protein A elution pH were especially good with all-prediction average errors of 0.2 and 1.6 pH unit, respectively, while predictions of some other properties were notably less good. This manuscript presents the events, methods, and results of the competition, and can serve as a tutorial and as a reference for in-house benchmarking by others. Organizations vary in their policies concerning disclosure of methods, but most managements were very cooperative with the Highland Games exercise, and considerable insight into common and best practices is available from the contributed methods. The accumulated data set will serve as a benchmarking tool for further development of in silico prediction tools.

Non-inferiority trials using a surrogate marker as the primary endpoint: An increasing phenotype in cardiovascular trials.

BACKGROUND/AIMS: Non-inferiority trials are increasing in cardiovascular medicine, with approval of many drugs and devices on the basis of such studies. Surrogate markers as primary endpoints have been also more frequently used for efficient assessment of cardiovascular interventions. However, there is uncertainty about their concordance with clinical outcomes. Non-inferiority design using a surrogate marker as a primary endpoint may pose particular challenges in clinical interpretation. We sought to explore the publication trends, methodology, and reporting features of non-inferiority cardiovascular trials that used a primary surrogate marker as the primary endpoint. METHODS: We searched six high-impact journals (The New England Journal of Medicine, The Journal of the American Medical Association, The Lancet, The Journal of the American College of Cardiology, Circulation, and European Heart Journal) from 1 January 1990 to 31 December 2018 and identified non-inferiority cardiovascular trials that used a surrogate marker as the primary endpoint. We assessed the non-inferiority margin reported in the manuscript and other publicly available platforms (e.g. protocol, clinicaltrials.gov). We also determined whether the included non-inferiority trials with surrogate markers as primary endpoints were followed by clinical outcome trials. RESULTS: We screened 15,553 publications and identified 247 cardiovascular trials that used a non-inferiority design. Of these, 37 had a surrogate marker as a primary endpoint (18 drug trials, 13 device trials, 6 others). All of these non-inferiority trials with surrogate outcomes were published after 2000, mostly in cardiology journals (13 in The Journal of the American College of Cardiology, 9 in European Heart Journal, 8 in Circulation, 6 in The Lancet, 1 in The New England Journal of Medicine), and their publication rate increased over time (p < 0.001 for linear trend). The median number of patients in the primary analysis was 300 (interquartile range: 202-465). The study protocol or a methods paper was publicly available for only 13 (35.1%) trials, of which the non-inferiority margin was not reported in 4 trials. In 16 studies (43.2%), the manuscript did not acknowledge the limitations of using a surrogate endpoint or the need for a definitive clinical outcome trial. Thirty-four trials (91.9%) concluded that the tested intervention met non-inferiority criteria. However, only five (13.5%) were followed by clinical outcomes trials the results of which did not always confirm non-inferiority. CONCLUSION: Non-inferiority trials that use a surrogate marker as the primary endpoint are being increasingly performed. However, these trials pose particular challenges with design, reporting, and interpretation, which are not systematically and consistently addressed or reported.

Automated High-Throughput Capillary Circular Dichroism and Intrinsic Fluorescence Spectroscopy for Rapid Determination of Protein Structure.

Assessing the physical stability of proteins is one of the most important challenges in the development, manufacture, and formulation of biotherapeutics. Here, we describe a method for combining and automating circular dichroism and intrinsic protein fluorescence spectroscopy. By robotically injecting samples from a 96-well plate into an optically compliant capillary flow cell, complementary information about the secondary and tertiary structural state of a protein can be collected in an unattended manner from considerably reduced volumes of sample compared to conventional techniques. We demonstrate the accuracy and reproducibility of this method. Furthermore, we show how structural screening can be used to monitor unfolding of proteins in two case studies using (i) a chaotropic denaturant (urea) and (ii) low-pH buffers used for monoclonal antibody (mAb) purification during Protein A chromatography.

Lymphovenous hemostasis and the role of platelets in regulating lymphatic flow and lymphatic vessel maturation.

Aside from the established role for platelets in regulating hemostasis and thrombosis, recent research has revealed a discrete role for platelets in the separation of the blood and lymphatic vascular systems. Platelets are activated by interaction with lymphatic endothelial cells at the lymphovenous junction, the site in the body where the lymphatic system drains into the blood vascular system, resulting in a platelet plug that, with the lymphovenous valve, prevents blood from entering the lymphatic circulation. This process, known as "lymphovenous hemostasis," is mediated by activation of platelet CLEC-2 receptors by the transmembrane ligand podoplanin expressed by lymphatic endothelial cells. Lymphovenous hemostasis is required for normal lymph flow, and mice deficient in lymphovenous hemostasis exhibit lymphedema and sometimes chylothorax phenotypes indicative of lymphatic insufficiency. Unexpectedly, the loss of lymph flow in these mice causes defects in maturation of collecting lymphatic vessels and lymphatic valve formation, uncovering an important role for fluid flow in driving endothelial cell signaling during development of collecting lymphatics. This article summarizes the current understanding of lymphovenous hemostasis and its effect on lymphatic vessel maturation and synthesizes the outstanding questions in the field, with relationship to human disease.

A systems approach to hemostasis: 4. How hemostatic thrombi limit the loss of plasma-borne molecules from the microvasculature.

Previous studies have shown that hemostatic thrombi formed in response to penetrating injuries have a core of densely packed, fibrin-associated platelets overlaid by a shell of less-activated, loosely packed platelets. Here we asked, first, how the diverse elements of this structure combine to stem the loss of plasma-borne molecules and, second, whether antiplatelet agents and anticoagulants that perturb thrombus structure affect the re-establishment of a tight vascular seal. The studies combined high-resolution intravital microscopy with a photo-activatable fluorescent albumin marker to simultaneously track thrombus formation and protein transport following injuries to mouse cremaster muscle venules. The results show that protein loss persists after red cell loss has ceased. Blocking platelet deposition with an αIIbß3antagonist delays vessel sealing and increases extravascular protein accumulation, as does either inhibiting adenosine 5'-diphosphate (ADP) P2Y12receptors or reducing integrin-dependent signaling and retraction. In contrast, sealing was unaffected by introducing hirudin to block fibrin accumulation or a Gi2α gain-of-function mutation to expand the thrombus shell. Collectively, these observations describe a novel approach for studying vessel sealing after injury in real time in vivo and show that (1) the core/shell architecture previously observed in arterioles also occurs in venules, (2) plasma leakage persists well beyond red cell escape and mature thrombus formation, (3) the most critical events for limiting plasma extravasation are the stable accumulation of platelets, ADP-dependent signaling, and the emergence of a densely packed core, not the accumulation of fibrin, and (4) drugs that affect platelet accumulation and packing can delay vessel sealing, permitting protein escape to continue.

Electrical Coupling and Synchronized Subthreshold Oscillations in the Inferior Olive of the Rhesus Macaque.

UNLABELLED: Inferior olive (IO) neurons are critical for motor coordination and exhibit oscillations in membrane potential that are subthreshold for spiking. The prevalence, coherence, and continuity of those subthreshold oscillations (STOs) depend upon resonant interactions between neighboring neurons supported by electrical coupling. Many studies of the olivocerebellar system in rodents, in which STOs were related to tremor, whisking, and licking, fueled a debate over whether IO STOs were relevant for primates whose repertoire of movement is generally less periodic. The debate was never well informed due to the lack of a direct examination of the physiological properties of primate IO neurons. Here, we obtained dual patch-clamp recordings of neighboring IO neurons from young adult macaques in brainstem slices and compared them to identical recordings from rats. Macaque IO neurons exhibited an equivalent prevalence of continuous STOs as rats (45 vs 54%, respectively). However, macaque STOs were slower (1-4 Hz) and did not overlap with the dominant 4-9 Hz frequency of rats. The slower STO frequency of macaques was at least partially due to a prolonged membrane time constant and increased membrane capacitance that could be attributed to stronger electrical coupling and greater total dendritic length. The presence of synchronized STOs in the IO of adult macaques, coincident with strong and prevalent electrical coupling, answers a fundamental outstanding question in cerebellar neuroscience and is consistent with a prominent role for synchronized oscillation in primate sensory-motor control. SIGNIFICANCE STATEMENT: It was debated whether inferior olive (IO) neurons of primates behave as synchronized oscillators as was found for rodents using intracellular, optical, and multielectrode recordings. An inability to resolve this issue using single-Purkinje cell extracellular recordings in monkeys limited our understanding of timing mechanisms in the primate brain. Using dual whole-cell recordings from the IO of young adult rhesus macaques in acutely prepared brainstem slices, our work demonstrates that pairs of primate IO neurons show synchronized oscillations in membrane potential. The findings have strong mechanistic and translational relevance, as IO activation has been implicated in humans' perceptual timing of sensory events and motricity.

Plumbagin improves the efficacy of androgen deprivation therapy in prostate cancer: A pre-clinical study.

BACKGROUND: Plumbagin is a candidate drug for the treatment of prostate cancer. Previous observations indicated that it may improve the efficacy of androgen deprivation therapy (ADT). This study evaluates the effectiveness of treatment with combinations of plumbagin and alternative strategies for ADT in mouse models of prostate cancer to support its clinical use. METHODS: Plumbagin was administered per oral in a new sesame oil formulation. Standard toxicology studies were performed in rats. For tumor growth studies, mouse prostate cancer cell spheroids were placed on top of grafted prostate tissue in a dorsal chamber and allowed to form tumors. Mice were separated in various treatment groups and tumor size was measured over time by intra-vital microscopy. Survival studies were done in mice after injection of prostate cancer cells in the prostate of male animals. Androgen receptor (AR) levels were analyzed by Western blot from prostate cancer cells treated with plumbagin. RESULTS: Plumbagin caused a decrease in AR levels in vitro. In mice, plumbagin at 1 mg/kg in sesame oil displayed low toxicity and caused a 50% tumor regression when combined with castration. The combination of plumbagin with various forms of chemical ADT including treatment with a GnRH receptor agonist, a GnRH receptor antagonist, or CYP17A1 inhibitors, outperformed ADT alone, increasing mouse survival compared to the standard regimen of castration alone. In contrast, the combination of plumbagin with AR antagonists, such as bicalutamide and enzalutamide, showed no improvement over AR antagonists alone. Thus, plumbagin is effective in combination with drugs that prevent the synthesis of testosterone or its conversion to dihydrotestosterone, but not with drugs that bind to AR. CONCLUSION: Plumbagin significantly improves the effect of ADT drugs currently used in the clinic, with few side effects in mice.

Production and stabilization of the trimeric influenza hemagglutinin stem domain for potentially broadly protective influenza vaccines.

The rapid dissemination of the 2009 pandemic H1N1 influenza virus emphasizes the need for universal influenza vaccines that would broadly protect against multiple mutated strains. Recent efforts have focused on the highly conserved hemagglutinin (HA) stem domain, which must undergo a significant conformational change for effective viral infection. Although the production of isolated domains of multimeric ectodomain proteins has proven difficult, we report a method to rapidly produce the properly folded HA stem domain protein from influenza virus A/California/05/2009 (H1N1) by using Escherichia coli-based cell-free protein synthesis and a simple refolding protocol. The T4 bacteriophage fibritin foldon placed at the C terminus of the HA stem domain induces trimer formation. Placing emphasis on newly exposed protein surfaces, several hydrophobic residues were mutated, two polypeptide segments were deleted, and the number of disulfide bonds in each monomer was reduced from four to two. High pH and Brij 35 detergent emerged as the most beneficial factors for improving the refolding yield. To stabilize the trimer of the HA stem-foldon fusion, new intermolecular disulfide bonds were finally introduced between foldon monomers and between stem domain monomers. The correct immunogenic conformation of the stabilized HA stem domain trimer was confirmed by using antibodies CR6261, C179, and FI6 that block influenza infection by binding to the HA stem domain trimer. These results suggest great promise for a broadly protective vaccine and also demonstrate a unique approach for producing individual domains of complex multimeric proteins.

Adherence to Methodological Standards in Research Using the National Inpatient Sample.

Importance: Publicly available data sets hold much potential, but their unique design may require specific analytic approaches. Objective: To determine adherence to appropriate research practices for a frequently used large public database, the National Inpatient Sample (NIS) of the Agency for Healthcare Research and Quality (AHRQ). Design, Setting, and Participants: In this observational study of the 1082 studies published using the NIS from January 2015 through December 2016, a representative sample of 120 studies was systematically evaluated for adherence to practices required by AHRQ for the design and conduct of research using the NIS. Exposures: None. Main Outcomes and Measures: All studies were evaluated on 7 required research practices based on AHRQ's recommendations and compiled under 3 domains: (1) data interpretation (interpreting data as hospitalization records rather than unique patients); (2) research design (avoiding use in performing state-, hospital-, and physician-level assessments where inappropriate; not using nonspecific administrative secondary diagnosis codes to study in-hospital events); and (3) data analysis (accounting for complex survey design of the NIS and changes in data structure over time). Results: Of 120 published studies, 85% (n = 102) did not adhere to 1 or more required practices and 62% (n = 74) did not adhere to 2 or more required practices. An estimated 925 (95% CI, 852-998) NIS publications did not adhere to 1 or more required practices and 696 (95% CI, 596-796) NIS publications did not adhere to 2 or more required practices. A total of 79 sampled studies (68.3% [95% CI, 59.3%-77.3%]) among the 1082 NIS studies screened for eligibility did not account for the effects of sampling error, clustering, and stratification; 62 (54.4% [95% CI, 44.7%-64.0%]) extrapolated nonspecific secondary diagnoses to infer in-hospital events; 45 (40.4% [95% CI, 30.9%-50.0%]) miscategorized hospitalizations as individual patients; 10 (7.1% [95% CI, 2.1%-12.1%]) performed state-level analyses; and 3 (2.9% [95% CI, 0.0%-6.2%]) reported physician-level volume estimates. Of 27 studies (weighted; 218 studies [95% CI, 134-303]) spanning periods of major changes in the data structure of the NIS, 21 (79.7% [95% CI, 62.5%-97.0%]) did not account for the changes. Among the 24 studies published in journals with an impact factor of 10 or greater, 16 (67%) did not adhere to 1 or more practices, and 9 (38%) did not adhere to 2 or more practices. Conclusions and Relevance: In this study of 120 recent publications that used data from the NIS, the majority did not adhere to required practices. Further research is needed to identify strategies to improve the quality of research using the NIS and assess whether there are similar problems with use of other publicly available data sets.

Threshold-based insulin-pump interruption for reduction of hypoglycemia.

BACKGROUND: The threshold-suspend feature of sensor-augmented insulin pumps is designed to minimize the risk of hypoglycemia by interrupting insulin delivery at a preset sensor glucose value. We evaluated sensor-augmented insulin-pump therapy with and without the threshold-suspend feature in patients with nocturnal hypoglycemia. METHODS: We randomly assigned patients with type 1 diabetes and documented nocturnal hypoglycemia to receive sensor-augmented insulin-pump therapy with or without the threshold-suspend feature for 3 months. The primary safety outcome was the change in the glycated hemoglobin level. The primary efficacy outcome was the area under the curve (AUC) for nocturnal hypoglycemic events. Two-hour threshold-suspend events were analyzed with respect to subsequent sensor glucose values. RESULTS: A total of 247 patients were randomly assigned to receive sensor-augmented insulin-pump therapy with the threshold-suspend feature (threshold-suspend group, 121 patients) or standard sensor-augmented insulin-pump therapy (control group, 126 patients). The changes in glycated hemoglobin values were similar in the two groups. The mean AUC for nocturnal hypoglycemic events was 37.5% lower in the threshold-suspend group than in the control group (980 ± 1200 mg per deciliter [54.4 ± 66.6 mmol per liter] × minutes vs. 1568 ± 1995 mg per deciliter [87.0 ± 110.7 mmol per liter] × minutes, P<0.001). Nocturnal hypoglycemic events occurred 31.8% less frequently in the threshold-suspend group than in the control group (1.5 ± 1.0 vs. 2.2 ± 1.3 per patient-week, P<0.001). The percentages of nocturnal sensor glucose values of less than 50 mg per deciliter (2.8 mmol per liter), 50 to less than 60 mg per deciliter (3.3 mmol per liter), and 60 to less than 70 mg per deciliter (3.9 mmol per liter) were significantly reduced in the threshold-suspend group (P<0.001 for each range). After 1438 instances at night in which the pump was stopped for 2 hours, the mean sensor glucose value was 92.6 ± 40.7 mg per deciliter (5.1 ± 2.3 mmol per liter). Four patients (all in the control group) had a severe hypoglycemic event; no patients had diabetic ketoacidosis. CONCLUSIONS: This study showed that over a 3-month period the use of sensor-augmented insulin-pump therapy with the threshold-suspend feature reduced nocturnal hypoglycemia, without increasing glycated hemoglobin values. (Funded by Medtronic MiniMed; ASPIRE ClinicalTrials.gov number, NCT01497938.).

A systems approach to hemostasis: 1. The interdependence of thrombus architecture and agonist movements in the gaps between platelets.

Hemostatic thrombi develop a characteristic architecture in which a core of highly activated platelets is covered by a shell of less-activated platelets. Here we have used a systems biology approach to examine the interrelationship of this architecture with transport rates and agonist distribution in the gaps between platelets. Studies were performed in mice using probes for platelet accumulation, packing density, and activation plus recently developed transport and thrombin activity probes. The results show that intrathrombus transport within the core is much slower than within the shell. The region of slowest transport coincides with the region of greatest packing density and thrombin activity, and appears prior to full platelet activation. Deleting the contact-dependent signaling molecule, Sema4D, delays platelet activation, but not the emergence of the low transport region. Collectively, these results suggest a timeline in which initial platelet accumulation and the narrowing gaps between platelets create a region of reduced transport that facilitates local thrombin accumulation and greater platelet activation, whereas faster transport rates within the shell help to limit thrombin accumulation and growth of the core. Thus, from a systems perspective, platelet accumulation produces an altered microenvironment that shapes thrombus architecture, which in turn affects agonist distribution and subsequent thrombus growth.

A systems approach to hemostasis: 2. Computational analysis of molecular transport in the thrombus microenvironment.

Hemostatic thrombi formed after a penetrating injury have a heterogeneous architecture in which a core of highly activated, densely packed platelets is covered by a shell of less-activated, loosely packed platelets. In the first manuscript in this series, we show that regional differences in intrathrombus protein transport rates emerge early in the hemostatic response and are preserved as the thrombus develops. Here, we use a theoretical approach to investigate this process and its impact on agonist distribution. The results suggest that hindered diffusion, rather than convection, is the dominant mechanism responsible for molecular movement within the thrombus. The analysis also suggests that the thrombus core, as compared with the shell, provides an environment for retaining soluble agonists such as thrombin, affecting the extent of platelet activation by establishing agonist-specific concentration gradients radiating from the site of injury. This analysis accounts for the observed weaker activation and relative instability of platelets in the shell and predicts that a failure to form a tightly packed thrombus core will limit thrombin accumulation, a prediction tested by analysis of data from mice with a defect in clot retraction.

A systems approach to hemostasis: 3. Thrombus consolidation regulates intrathrombus solute transport and local thrombin activity.

Hemostatic thrombi formed after a penetrating injury have a distinctive structure in which a core of highly activated, closely packed platelets is covered by a shell of less-activated, loosely packed platelets. We have shown that differences in intrathrombus molecular transport emerge in parallel with regional differences in platelet packing density and predicted that these differences affect thrombus growth and stability. Here we test that prediction in a mouse vascular injury model. The studies use a novel method for measuring thrombus contraction in vivo and a previously characterized mouse line with a defect in integrin αIIbß3 outside-in signaling that affects clot retraction ex vivo. The results show that the mutant mice have a defect in thrombus consolidation following vascular injury, resulting in an increase in intrathrombus transport rates and, as predicted by computational modeling, a decrease in thrombin activity and platelet activation in the thrombus core. Collectively, these data (1) demonstrate that in addition to the activation state of individual platelets, the physical properties of the accumulated mass of adherent platelets is critical in determining intrathrombus agonist distribution and platelet activation and (2) define a novel role for integrin signaling in the regulation of intrathrombus transport rates and localization of thrombin activity.

High throughput chromatography strategies for potential use in the formal process characterization of a monoclonal antibody.

High throughput experimental strategies are central to the rapid optimization of biologics purification processes. In this work, we extend common high throughput technologies towards the characterization of a multi-column chromatography process for a monoclonal antibody (mAb). Scale-down strategies were first evaluated by comparing breakthrough, retention, and performance (yields and clearance of aggregates and host cell protein) across miniature and lab scale columns. The process operating space was then evaluated using several integrated formats, with batch experimentation to define process testing ranges, miniature columns to evaluate the operating space, and comparison to traditional scale columns to establish scale-up correlations and verify the determined operating space. When compared to an independent characterization study at traditional lab column scale, the high throughput approach identified the same control parameters and similar process sensitivity. Importantly, the high throughput approach significantly decreased time and material needs while improving prediction robustness. Miniature columns and manufacturing scale centerpoint data comparisons support the validity of this approach, making the high throughput strategy an attractive and appropriate scale-down tool for the formal characterization of biotherapeutic processes in the future if regulatory acceptance of the miniature column data can be achieved. Biotechnol. Bioeng. 2016;113: 1273-1283. © 2015 Wiley Periodicals, Inc.