Implementation of an end-to-end continuous bioprocessing platform using novel technologies

One significant opportunity for evolutionary change in the biopharmaceutical industry is the widespread adoption of integrated continuous bioprocessing for biologics manufacturing. Key to its success is the availability of novel upstream and downstream technologies that will not only reduce facility footprint, capital expenses and product cost of goods (CoGs), but also will increase process productivity, flexibility and further facilitate the utilization of single-use and/or disposable technologies. In this context, the suite of cutting-edge technologies we have evaluated to enable cost effective and reliable implementation of continuous bioprocessing of biological drugs, included the Cadence™ Acoustic Separator exploiting acoustic wave separation technology (AWS), Cadence Inline Concentrators within the single-pass TFF (SPTFF) platform, the Cadence BioSMB PD multicolumn continuous chromatography platform using a KANEKA KanCap A™ based platform and novel continuous diafiltration strategies, to address the innovation gap to provide a simplified solution for the continuous final formulation step. By utilizing a 20L CHO fed-batch cell culture bioreactor with cell density range of 25x106 – 30x106 cells/mL and 65 to 90% cell viability, multiple in-house feasibility runs were conducted through a novel integrated continuous bioprocessing train of unit operations. For instance, while achieving ≥90% continuous clarification yield for the processing of a batch with 1.25 g/L titer, 25x106 cells/mL & ~70% viability this new process platform was able to deliver ≥ 2 g/h mAb for the continuous purification train utilizing a stable 4-fold continuous concentration step for the integration of continuous clarification and continuous capture trains. We further intensified the process and by running over a 24h period we were able to purify in excess of 100g mAb over this period giving a productivity of this integrated system of ~124g mAb per day. This was carried out in a dedicated Continuous BioProcessing facility within a footprint of just 36m2. With the coupling of the novel continuous clarification, continuous capture, continuous virus inactivation, continuous polishing, continuous viral clearance and continuous final formulation steps, in this platform, using current PD-scale bioreactors, we have demonstrated end-to-end continuous biomanufacture that will generate 1-5 g/h mAb. This presentation will provide a risk-based and data-driven overview of an integrated continuous bioprocessing platform and highlight the requirements, challenges and opportunities for product development, process monitoring, validati

The Calibration of the HST Kuiper Belt Object Search: Setting the Record Straight

The limiting magnitude of the HST data set used by Cochran et al. (1995) to detect small objects in the Kuiper belt is reevaluated, and the methods used are described in detail. It is shown, by implanting artificial objects in the original HST images, and re-reducing the images using our original algorithm, that the limiting magnitude of our images (as defined by the 50% detectability limit) is $V=28.4$. This value is statistically the same as the value found in the original analysis. We find that $\sim50$ of the moving Kuiper belt objects with $V=27.9$ are detected when trailing losses are included. In the same data in which these faint objects are detected, we find that the number of false detections brighter than $V=28.8$ is less than one per WFPC2 image. We show that, primarily due to a zero-point calibration error, but partly due to inadequacies in modeling the HST'S data noise characteristics and Cochran et al.'s reduction techniques, Brown et al. 1997 underestimate the SNR of objects in the HST dataset by over a factor of 2, and their conclusions are therefore invalid.Comment: Accepted to ApJ Letters; 10 pages plus 3 figures, LaTe

Acoustic Wave Separation – A non-filtration approach for continuous clarification of perfusion cell culture prior to capture chromatography

Advances in perfusion cell culture have led to cell densities in excess of 100 million cells/mL with product titers similar to those obtained in fed batch (3-5 g/L). This performance has necessitated improvements in the yield and efficiency of the cell harvest and clarification stage to generate a stream of Harvested Cell Culture Fluid (HCCF) for capture chromatography and subsequent downstream processing. This is further driven by the evolution of continuous processes where there is a preference for a continuous feed of HCCF available for direct load to the continuous multicolumn capture chromatography step. In the present work we report on a novel disruptive and scalable single-use technology for cell retention during perfusion cell culture based on an acoustophoretic separation. Acoustic Wave Separation (AWS) technology exploits the use of low frequency acoustic forces to generate a three-dimensional standing wave across a flow channel. Recirculating cell culture from a perfusion bioreactor enters the flow channel and passes below the acoustic zone. The product-containing stream of HCCF is removed from the recirculating cell culture by passage through the acoustic zone. This yields a well clarified HCCF that can be polished using a small area filter. We report the continuous cell retention during a perfusion culture of a CHO cell line expressing a mAb. At process development (PD) scale we demonstrate the ability to continuously process CHO cell culture and retain cells at densities of up to 100 million cells/mL, at flow rates of up to 2 bioreactor volumes per day. Since the clarification technology does not involve the use of hollow fiber tangential flow filtration (TFF) we ensure 100% transmission of the mAb through the AWS device. The closed system remains operational for up to 60 days enabling this scalable technology to be suitable for use in clinical manufacture. The post-AWS HCCF is 99% clarified and any residual cellular material can be removed using a small gamma stable membrane filter or directly loaded onto a 0.2 micron filter prior to chromatography. Additionally, no demonstrable adverse effects have been identified for the quality of the HCCF, the product itself, or the viability of the returning perfusion cell culture following cell retention using AWS technology. AWS technology enables the continuous cell retention from recirculating cell culture withdrawn from perfusion bioreactors in a single-use operation. AWS technology has been shown to perform well at cell densities of up to 100 million cells/mL, so is well positioned to meet the cell retention requirements of emerging higher cell density perfusion processes that are gaining momentum in the biotech space. This novel cell retention approach offers economic benefits in terms of yield improvement as well as eliminating the hollow fiber TFF operation. This offers the advantage of a stable mAb concentration in the HCCF stream during the perfusion process. This facilitates improved process control since the volume of HCCF to load on to the capture columns remains constant which is especially important during continuous multicolumn chromatography. By comparison with hollow fiber TFF, the mAb concentration varies during the cell retention process making an integrated process more complex to control

The Formation of Uranus and Neptune in Solid-Rich Feeding Zones: Connecting Chemistry and Dynamics

The core accretion theory of planet formation has at least two fundamental problems explaining the origins of Uranus and Neptune: (1) dynamical times in the trans-Saturnian solar nebula are so long that core growth can take > 15 Myr, and (2) the onset of runaway gas accretion that begins when cores reach 10 Earth masses necessitates a sudden gas accretion cutoff just as the ice giant cores reach critical mass. Both problems may be resolved by allowing the ice giants to migrate outward after their formation in solid-rich feeding zones with planetesimal surface densities well above the minimum-mass solar nebula. We present new simulations of the formation of Uranus and Neptune in the solid-rich disk of Dodson-Robinson et al. (2009) using the initial semimajor axis distribution of the Nice model (Gomes et al. 2005; Morbidelli et al. 2005; Tsiganis et al. 2005), with one ice giant forming at 12 AU and the other at 15 AU. The innermost ice giant reaches its present mass after 3.8-4.0 Myr and the outermost after 5.3-6 Myr, a considerable time decrease from previous one-dimensional simulations (e.g. Pollack et al. 1996). The core masses stay subcritical, eliminating the need for a sudden gas accretion cutoff. Our calculated carbon mass fractions of 22% are in excellent agreement with the ice giant interior models of Podolak et al. (1995) and Marley et al. (1995). Based on the requirement that the ice giant-forming planetesimals contain >10% mass fractions of methane ice, we can reject any solar system formation model that initially places Uranus and Neptune inside the orbit of Saturn. We also demonstrate that a large population of planetesimals must be present in both ice giant feeding zones throughout the lifetime of the gaseous nebula.Comment: Accepted for publication in Icarus. 9 pages, including 3 figure

Qualitative assessment of attitudes and knowledge on preterm birth in Malawi and within country framework of care

BACKGROUND: The overarching goal of this study was to qualitatively assess baseline knowledge and perceptions regarding preterm birth (PTB) and oral health in an at-risk, low resource setting surrounding Lilongwe, Malawi. The aims were to determine what is understood regarding normal length of gestation and how gestational age is estimated, to identify common language for preterm birth, and to assess what is understood as options for PTB management. As prior qualitative research had largely focused on patient or client-based focused groups, we primarily focused on groups comprised of community health workers (CHWs) and providers. METHODS: A qualitative study using focus-group discussions, incidence narrative, and informant interviews amongst voluntary participants. Six focus groups were comprised of CHWs, patient couples, midwives, and clinical officers (n = 33) at two rural health centers referring to Kamuzu Central Hospital. Semi-structured questions facilitated discussion of PTB and oral health (inclusive of periodontal disease), including definitions, perception, causation, management, and accepted interventions. RESULTS: Every participant knew of women who had experienced “a baby born too soon”, or preterm birth. All participants recognized both an etiology conceptualization and disease framework for preterm birth, distinguished PTB from miscarriage and macerated stillbirth, and articulated a willingness to engage in studies aimed at prevention or management. Identified gaps included: (1) discordance in the definition of PTB (i.e., 28–34 weeks or less than the 8(th) month, but with a corresponding fetal weight ranging 500 to 2300 grams); (2) utility and regional availability of antenatal steroids for prevention of preterm infant morbidity and mortality; (3) need for antenatal referral for at-risk women, or with symptoms of preterm birth. There was no evident preference for route of progesterone for the prevention of recurrent PTB. CONCLUSIONS: Qualitative research was useful in (1) identifying gaps in knowledge in urban and rural Malawi, and (2) informing the development of educational materials and implementation of programs or trials ultimately aimed at reducing PTB. As a result of this qualitative work, implementation planning was focused on the gaps in knowledge, dissemination of knowledge (to both patients and providers), and practical solutions to barriers in known efficacious therapies

Does the motor system need intermittent control?

Explanation of motor control is dominated by continuous neurophysiological pathways (e.g. trans-cortical, spinal) and the continuous control paradigm. Using new theoretical development, methodology and evidence, we propose intermittent control, which incorporates a serial ballistic process within the main feedback loop, provides a more general and more accurate paradigm necessary to explain attributes highly advantageous for competitive survival and performance

Prediction of marbofloxacin dosage for the pig pneumonia pathogens Actinobacillus pleuropneumoniae and Pasteurella multocida by pharmacokinetic/pharmacodynamic modelling

PK/PD integration data. (DOCX 7 kb

A chemical survey of exoplanets with ARIEL

Thousands of exoplanets have now been discovered with a huge range of masses, sizes and orbits: from rocky Earth-like planets to large gas giants grazing the surface of their host star. However, the essential nature of these exoplanets remains largely mysterious: there is no known, discernible pattern linking the presence, size, or orbital parameters of a planet to the nature of its parent star. We have little idea whether the chemistry of a planet is linked to its formation environment, or whether the type of host star drives the physics and chemistry of the planet’s birth, and evolution. ARIEL was conceived to observe a large number (~1000) of transiting planets for statistical understanding, including gas giants, Neptunes, super-Earths and Earth-size planets around a range of host star types using transit spectroscopy in the 1.25–7.8 μm spectral range and multiple narrow-band photometry in the optical. ARIEL will focus on warm and hot planets to take advantage of their well-mixed atmospheres which should show minimal condensation and sequestration of high-Z materials compared to their colder Solar System siblings. Said warm and hot atmospheres are expected to be more representative of the planetary bulk composition. Observations of these warm/hot exoplanets, and in particular of their elemental composition (especially C, O, N, S, Si), will allow the understanding of the early stages of planetary and atmospheric formation during the nebular phase and the following few million years. ARIEL will thus provide a representative picture of the chemical nature of the exoplanets and relate this directly to the type and chemical environment of the host star. ARIEL is designed as a dedicated survey mission for combined-light spectroscopy, capable of observing a large and well-defined planet sample within its 4-year mission lifetime. Transit, eclipse and phase-curve spectroscopy methods, whereby the signal from the star and planet are differentiated using knowledge of the planetary ephemerides, allow us to measure atmospheric signals from the planet at levels of 10–100 part per million (ppm) relative to the star and, given the bright nature of targets, also allows more sophisticated techniques, such as eclipse mapping, to give a deeper insight into the nature of the atmosphere. These types of observations require a stable payload and satellite platform with broad, instantaneous wavelength coverage to detect many molecular species, probe the thermal structure, identify clouds and monitor the stellar activity. The wavelength range proposed covers all the expected major atmospheric gases from e.g. H2O, CO2, CH4 NH3, HCN, H2S through to the more exotic metallic compounds, such as TiO, VO, and condensed species. Simulations of ARIEL performance in conducting exoplanet surveys have been performed – using conservative estimates of mission performance and a full model of all significant noise sources in the measurement – using a list of potential ARIEL targets that incorporates the latest available exoplanet statistics. The conclusion at the end of the Phase A study, is that ARIEL – in line with the stated mission objectives – will be able to observe about 1000 exoplanets depending on the details of the adopted survey strategy, thus confirming the feasibility of the main science objectives.Peer reviewedFinal Published versio

Cometary Origin of the Zodiacal Cloud and Carbonaceous Micrometeorites

The zodiacal cloud is a thick circumsolar disk of small debris particles produced by asteroid collisions and comets. Here, we present a zodiacal cloud model based on the orbital properties and lifetimes of comets and asteroids, and on the dynamical evolution of dust after ejection. The model is quantitatively constrained by IRAS observations of thermal emission, but also qualitatively consistent with other zodiacal cloud observations. We find that 85-95% of the observed mid-infrared emission is produced by particles from the Jupiter-family comets (JFCs) and $<$10% by dust from long period comets. Asteroidal dust is found to be present at $<$10%. We suggest that spontaneous disruptions of JFCs, rather than the usual cometary activity driven by sublimating volatiles, is the main mechanism that librates cometary particles into the zodiacal cloud. Our results imply that JFC particles represent $\sim$85% of the total mass influx at Earth. Since their atmospheric entry speeds are typically low ($\approx$14.5 km s$^{-1}$ mean for D=100-200 $\mu$m with $\approx$12 km s$^{-1}$ being the most common case), many JFC grains should survive frictional heating and land on the Earth's surface. This explains why most micrometeorites collected in antarctic ice have primitive carbonaceous composition. The present mass of the inner zodiacal cloud at $<$5 AU is estimated to be 1-$2\times10^{19}$ g, mainly in D=100-200 $\mu$m particles. The inner zodiacal cloud should have been $>10^4$ times brighter during the Late Heavy Bombardment (LHB) epoch $\approx$3.8 Gyr ago, when the outer planets scattered numerous comets into the inner solar system. The bright debris disks with a large 24-$\mu$m excess observed around mature stars may be an indication of massive cometary populations existing in those systems

Periodontal disease and atherosclerotic vascular disease: Does the evidence support an independent association?: A scientific statement from the American heart association

A link between oral health and cardiovascular disease has been proposed for more than a century. Recently, concern about possible links between periodontal disease (PD) and atherosclerotic vascular disease (ASVD) has intensified and is driving an active field of investigation into possible association and causality. The 2 disorders share several common risk factors, including cigarette smoking, age, and diabetes mellitus. Patients and providers are increasingly presented with claims that PD treatment strategies offer ASVD protection; these claims are often endorsed by professional and industrial stakeholders. The focus of this review is to assess whether available data support an independent association between ASVD and PD and whether PD treatment might modify ASVD risks or outcomes. It also presents mechanistic details of both PD and ASVD relevant to this topic. The correlation of PD with ASVD outcomes and surrogate markers is discussed, as well as the correlation of response to PD therapy with ASVD event rates. Methodological issues that complicate studies of this association are outlined, with an emphasis on the terms and metrics that would be applicable in future studies. Observational studies to date support an association between PD and ASVD independent of known confounders. They do not, however, support a causative relationship. Although periodontal interventions result in a reduction in systemic inflammation and endothelial dysfunction in short-term studies, there is no evidence that they prevent ASVD or modify its outcomes