An assessment of the impact of Raman based glucose feedback control on CHO cell bioreactor process development.

Process analytical technology (PAT) tools such as Raman Spectroscopy have become established tools for real time measurement of CHO cell bioreactor process variables and are aligned with the QbD approach to manufacturing. These tools can have a significant impact on process development if adopted early, creating an end-to-end PAT/QbD focused process. This study assessed the impact of Raman based feedback control on early and late phase development bioreactors by using a Raman based PLS model and PAT management system to control glucose in two CHO cell line bioreactor processes. The impact was then compared to bioreactor processes which used manual bolus fed methods for glucose feed delivery. Process improvements were observed in terms of overall bioreactor health, product output and product quality. Raman controlled batches for Cell Line 1 showed a reduction in glycation of 43.4% and 57.9%, respectively. Cell Line 2 batches with Raman based feedback control showed an improved growth profile with higher VCD and viability and a resulting 25% increase in overall product titer with an improved glycation profile. The results presented here demonstrate that Raman spectroscopy can be used in both early and late-stage process development and design for consistent and controlled glucose feed delivery.

SARM1 Promotes Photoreceptor Degeneration in an Oxidative Stress Model of Retinal Degeneration.

SARM1 (sterile alpha and armadillo motif-containing protein) is a highly conserved Toll/IL-1 Receptor (TIR) adaptor with important roles in mediating immune responses. Studies in the brain have shown that SARM1 plays a role in induction of neuronal axon degeneration in response to a variety of injuries. We recently demonstrated that SARM1 is pro-degenerative in a genetic model of inherited retinopathy. This current study aimed to characterise the effect of SARM1 deletion in an alternative model of retinal degeneration (RD) in which the retinal pigment epithelium (RPE) fragments following administration of oxidising agent, sodium iodate (NaIO3), leading to subsequent photoreceptor cell death. Following administration of NaIO3, we observed no apparent difference in rate of loss of RPE integrity in SARM1 deficient mice compared to WT counterparts. However, despite no differences in RPE degeneration, photoreceptor cell number and retinal thickness were increased in Sarm1-/- mice compared to WT counterparts. This apparent protection of the photoreceptors in SARM1 deficient mice is supported by an observed decrease in pro-apoptotic caspase-3 in the photoreceptor layer of Sarm1-/- mice compared to WT. Together these data indicate a pro-degenerative role for SARM1 in the photoreceptors, but not in the RPE, in an oxidative stress induced model of retinal degeneration consistent with its known degenerative role in neurons in a range of neurodegenerative settings.

Raman based chemometric model development for glycation and glycosylation real time monitoring in a manufacturing scale CHO cell bioreactor process.

The Quality by Design (QbD) approach to the production of therapeutic monoclonal antibodies (mAbs) emphasizes an understanding of the production process ensuring product quality is maintained throughout. Current methods for measuring critical quality attributes (CQAs) such as glycation and glycosylation are time and resource intensive, often, only tested offline once per batch process. Process analytical technology (PAT) tools such as Raman spectroscopy combined with chemometric modeling can provide real time measurements process variables and are aligned with the QbD approach. This study utilizes these tools to build partial least squares (PLS) regression models to provide real time monitoring of glycation and glycosylation profiles. In total, seven cell line specific chemometric PLS models; % mono-glycated, % non-glycated, % G0F-GlcNac, % G0, % G0F, % G1F, and % G2F were considered. PLS models were initially developed using small scale data to verify the capability of Raman to measure these CQAs effectively. Accurate PLS model predictions were observed at small scale (5 L). At manufacturing scale (2000 L) some glycosylation models showed higher error, indicating that scale may be a key consideration in glycosylation profile PLS model development. Model robustness was then considered by supplementing models with a single batch of manufacturing scale data. This data addition had a significant impact on the predictive capability of each model, with an improvement of 77.5% in the case of the G2F. The finalized models show the capability of Raman as a PAT tool to deliver real time monitoring of glycation and glycosylation profiles at manufacturing scale.

Clinical Outcomes and Inflammatory Responses of the Frequent Exacerbator in Pulmonary Rehabilitation: A Prospective Cohort Study.

Frequent exacerbators of Chronic Obstructive Pulmonary Disease (COPD) is a distinct clinical phenotype characterised by systemic inflammation. Study objectives were to determine clinical outcomes of pulmonary rehabilitation in frequent exacerbators and the impact this has on the key surrogate markers of this phenotype. Eighty-five mild-very severe COPD patients (FEV1 pred, 52 ± 18%) were categorised as frequent (≥2 exacerbations per year, n = 50) or infrequent exacerbators (≤1 exacerbation per year, n = 35). The primary outcomes were completion rates of pulmonary rehabilitation (clinical) and plasma fibrinogen (biological). Secondary outcomes were: incremental shuttle (ISWT) & endurance shuttle walk tests (ESWT), chronic respiratory disease questionnaire (CRQ), hospital anxiety and depression scale (HADS), plasma C-reactive protein (CRP), blood leukocyte counts, blood neutrophil activation (CD11b, CD62L, CD66b) and subsets (mature, immature, suppressive, progenitor). Fibrinogen and CRP concentrations were determined via ELISA's with neutrophil activation markers assessed using flow cytometry. Frequent exacerbators were less likely to complete pulmonary rehabilitation (44% vs 69%; p = 0.025). Both groups experienced improvements in ISWT (p < 0.001), ESWT (p < 0.001), all domains of the CRQ (p < 0.001) and Depression (p = 0.017). Pulmonary rehabilitation reduced resting concentrations of fibrinogen (frequent exacerbators = 12%, infrequent exacerbators = 4%, p = 0.033) and % of progenitor blood neutrophils (p = 0.015) in both groups, with reductions in total blood leukocyte (p = 0.018) and neutrophil counts (p = 0.018) also observed in frequent exacerbators. No significant reductions in CRP concentration (p = 0.937), neutrophil activation (CD11b, p = 0.553; CD62L, p = 0.070; CD66b, p = 0.317), or other neutrophil subsets (mature, p = 0.313; immature, p = 0.756; suppressive, p = 0.259) were observed. Frequent exacerbators of COPD were less likely to complete pulmonary rehabilitation, but those who complete experience similar benefits to infrequent exacerbators. Pulmonary rehabilitation may serve to have immune-modulatory properties for frequent exacerbators.

SARM1 deficiency promotes rod and cone photoreceptor cell survival in a model of retinal degeneration.

Retinal degeneration is the leading cause of incurable blindness worldwide and is characterised by progressive loss of light-sensing photoreceptors in the neural retina. SARM1 is known for its role in axonal degeneration, but a role for SARM1 in photoreceptor cell degeneration has not been reported. SARM1 is known to mediate neuronal cell degeneration through depletion of essential metabolite NAD and induction of energy crisis. Here, we demonstrate that SARM1 is expressed in photoreceptors, and using retinal tissue explant, we confirm that activation of SARM1 causes destruction of NAD pools in the photoreceptor layer. Through generation of rho -/- sarm1 -/- double knockout mice, we demonstrate that genetic deletion of SARM1 promotes both rod and cone photoreceptor cell survival in the rhodopsin knockout (rho -/- ) mouse model of photoreceptor degeneration. Finally, we demonstrate that SARM1 deficiency preserves cone visual function in the surviving photoreceptors when assayed by electroretinography. Overall, our data indicate that endogenous SARM1 has the capacity to consume NAD in photoreceptor cells and identifies a previously unappreciated role for SARM1-dependent cell death in photoreceptor cell degeneration.

New insights into subsurface imaging of carbon nanotubes in polymer composites via scanning electron microscopy.

Despite many studies of subsurface imaging of carbon nanotube (CNT)-polymer composites via scanning electron microscopy (SEM), significant controversy exists concerning the imaging depth and contrast mechanisms. We studied CNT-polyimide composites and, by three-dimensional reconstructions of captured stereo-pair images, determined that the maximum SEM imaging depth was typically hundreds of nanometers. The contrast mechanisms were investigated over a broad range of beam accelerating voltages from 0.3 to 30 kV, and ascribed to modulation by embedded CNTs of the effective secondary electron (SE) emission yield at the polymer surface. This modulation of the SE yield is due to non-uniform surface potential distribution resulting from current flows due to leakage and electron beam induced current. The importance of an external electric field on SEM subsurface imaging was also demonstrated. The insights gained from this study can be generally applied to SEM nondestructive subsurface imaging of conducting nanostructures embedded in dielectric matrices such as graphene-polymer composites, silicon-based single electron transistors, high resolution SEM overlay metrology or e-beam lithography, and have significant implications in nanotechnology.

Payment by results in forensic mental health.

Forensic mental health services are low-volume, high-cost services. Payment by results (PbR) is the UK s latest attempt to improve efficiency and controls pending behaviours within the secure services. This article discusses the utility of the PbR mechanic in forensic mental health. It explores PbR implementation in non-forensic mental health settings, similar funding processes internationally, and early PbR implementation work in the UK's secure services. Finally, the article discusses the challenges faced when implementing PbR in forensic mental health services and puts forward possible next steps in determining the utility of PbR in forensic mental health.

A quantitative assessment of carbon nanotube dispersion in polymer matrices.

Quantifying the nature and extent of the dispersion of nanomaterials in polymer matrices is the important first step in understanding the relationship between the nanoscale structure and the bulk scale functional performance of nanocomposites. We present here a methodology for using scanning electron microscope images of nanocomposites taken under high accelerating voltages to quantify four parameters that relate to the dispersion of the nanomaterial. This image analysis methodology is general and applicable to images from other microscopes as well. The analysis performed here was done on representative local areas of six samples to determine the effects of processing conditions, matrix chemistry, and carbon nanotube composition on the level of dispersion. Future work will involve expanding this analysis to rapidly cover larger areas and reducing the data in a manner that is similar to the approach of small angle scattering studies.