Processing and formulation insights for designing quality into lyophilised biopharmaceuticals

This thesis makes an original contribution to the field of formulation development by providing new experimental data and insights into the effect of processing and formulation conditions on the quality of lyophilised biopharmaceuticals. The quality attributes of lyophilised products include: a quick reconstitution time, product elegance and protein stability, which are known to be affected by processing and formulation parameters. However, choice of formulation excipients or processing conditions often relies on previous experience rather than mechanistic insight. The motivation of this thesis was therefore to provide a greater understanding of how process variables andexcipient choice affect these quality attributes. Bovine serum albumin (BSA) and immunoglobulin G (IgG) were used as model proteins to investigate formulation conditions, which included the excipient, the lyophilisation cooling profile and duration of the annealing step. BSA was also used as a model protein to explore the effects of sucrose and arginine as lyoprotectants. Unique to this study was the investigation of arginine salts as lyoprotectants, wherein the counterions were dicarboxylic acids with increasing chain length. Two key results regarding quality attributes were observed. Firstly, characterisation of the lyophilised structure established that there was an optimal annealing time, beyond which there was an increase in primary drying time, batch heterogeneity and variable moisture content. Secondly, a relationship was found between decreasing dicarboxylic acid chain length and improved protein stability. To explain these findings, two mechanisms are proposed that account for ice crystal growth during annealing and the observed changes in protein stability at the molecular level. Significantly, this research provides insights for future formulation development studies

National Evaluation of the Partnerships for Older People Projects: Interim Report of Progress

This second interim report provides a summary of key findings from the National Evaluation of the Department of Health’s POPP Programme. These summary findings are based on data collected and analysed over the last two years of the POPP programme (April 2006 to March 2008) and are made available to support emerging learning around prevention and early intervention. As the majority of the pilot sites still have one year to run, these findings, outcomes and subsequent discussion may be subject to change. All the issues and evidence on which these findings are based will be made available in the Final Report of the National Evaluation to be published in Autumn 2009

Selenium digestibility and bioactivity in dogs : what the can can, the kibble can't

There is a growing concern for the long-term health effects of selenium (Se) over-or underfeeding. The efficiency of utilization of dietary Se is subject to many factors. Our study in dogs evaluated the effect of diet type (canned versus kibble) and dietary protein concentration on Se digestibility and bioactivity. Canned and kibble diets are commonly used formats of dog food, widely ranging in protein concentration. Twenty-four Labrador retrievers were used and four canned and four kibble diets were selected with crude protein concentrations ranging from 10.1 to 27.5 g/MJ. Crude protein concentration had no influence on the digestibility of Se in either canned or kibble diets, but a lower Se digestibility was observed in canned compared to kibble diets. However, the biological activity of Se, as measured by whole blood glutathione peroxidase, was higher in dogs fed the canned diets than in dogs fed the kibble diets and decreased with increasing crude protein intake. These results indicate that selenium recommendations in dog foods need to take diet type into account

Individualised profiling of white matter organisation in moderate-to-severe traumatic brain injury patients

Background and purpose Approximately 65% of moderate-to-severe traumatic brain injury (m-sTBI) patients present with poor long-term behavioural outcomes, which can significantly impair activities of daily living. Numerous diffusion-weighted MRI studies have linked these poor outcomes to decreased white matter integrity of several commissural tracts, association fibres and projection fibres in the brain. However, most studies have focused on group-based analyses, which are unable to deal with the substantial between-patient heterogeneity in m-sTBI. As a result, there is increasing interest and need in conducting individualised neuroimaging analyses. Materials and methods Here, we generated a detailed subject-specific characterisation of microstructural organisation of white matter tracts in 5 chronic patients with m-sTBI (29 – 49y, 2 females), presented as a proof-of-concept. We developed an imaging analysis framework using fixel-based analysis and TractLearn to determine whether the values of fibre density of white matter tracts at the individual patient level deviate from the healthy control group (n = 12, 8F, Mage = 35.7y, age range 25 – 64y). Results Our individualised analysis revealed unique white matter profiles, confirming the heterogenous nature of m-sTBI and the need of individualised profiles to properly characterise the extent of injury. Future studies incorporating clinical data, as well as utilising larger reference samples and examining the test–retest reliability of the fixel-wise metrics are warranted. Conclusions Individualised profiles may assist clinicians in tracking recovery and planning personalised training programs for chronic m-sTBI patients, which is necessary to achieve optimal behavioural outcomes and improved quality of life

Potent suppression of vascular smooth muscle cell migration and human neointimal hyperplasia by KV1.3 channel blockers

Aim - The aim of the study was to determine the potential for KV1 potassium channel blockers as inhibitors of human neoinitimal hyperplasia. Methods and results - Blood vessels were obtained from patients or mice and studied in culture. Reverse transcriptasepolymerase chain reaction and immunocytochemistry were used to detect gene expression. Whole-cell patch-clamp, intracellular calcium measurement, cell migration assays, and organ culture were used to assess channel function.  KV1.3 was unique among the  KV1 channels in showing preserved and up-regulated expression when the vascular smooth muscle cells switched to the proliferating phenotype. There was strong expression in neointimal formations. Voltage-dependent potassium current in proliferating cells was sensitive to three different blockers of  KV1.3 channels. Calcium entry was also inhibited. All three blockers reduced vascular smooth muscle cell migration and the effects were non-additive. One of the blockers (margatoxin) was highly potent, suppressing cell migration with an IC of 85 pM. Two of the blockers were tested in organ-cultured human vein samples and both inhibited neointimal hyperplasia. Conclusion - KV1.3 potassium channels are functional in proliferating mouse and human vascular smooth muscle cells and have positive effects on cell migration. Blockers of the channels may be useful as inhibitors of neointimal hyperplasia and other unwanted vascular remodelling events

Annotation of two large contiguous regions from the Haemonchus contortus genome using RNA-seq and comparative analysis with Caenorhabditis elegans

The genomes of numerous parasitic nematodes are currently being sequenced, but their complexity and size, together with high levels of intra-specific sequence variation and a lack of reference genomes, makes their assembly and annotation a challenging task. Haemonchus contortus is an economically significant parasite of livestock that is widely used for basic research as well as for vaccine development and drug discovery. It is one of many medically and economically important parasites within the strongylid nematode group. This group of parasites has the closest phylogenetic relationship with the model organism Caenorhabditis elegans, making comparative analysis a potentially powerful tool for genome annotation and functional studies. To investigate this hypothesis, we sequenced two contiguous fragments from the H. contortus genome and undertook detailed annotation and comparative analysis with C. elegans. The adult H. contortus transcriptome was sequenced using an Illumina platform and RNA-seq was used to annotate a 409 kb overlapping BAC tiling path relating to the X chromosome and a 181 kb BAC insert relating to chromosome I. In total, 40 genes and 12 putative transposable elements were identified. 97.5% of the annotated genes had detectable homologues in C. elegans of which 60% had putative orthologues, significantly higher than previous analyses based on EST analysis. Gene density appears to be less in H. contortus than in C. elegans, with annotated H. contortus genes being an average of two-to-three times larger than their putative C. elegans orthologues due to a greater intron number and size. Synteny appears high but gene order is generally poorly conserved, although areas of conserved microsynteny are apparent. C. elegans operons appear to be partially conserved in H. contortus. Our findings suggest that a combination of RNA-seq and comparative analysis with C. elegans is a powerful approach for the annotation and analysis of strongylid nematode genomes

Insights into the influence of the cooling profile on the reconstitution times of amorphous lyophilized protein formulations

Lyophilized protein formulations must be reconstituted back into solution prior to patient administration and in this regard long reconstitution times are not ideal. The factors that govern reconstitution time remain poorly understood. The aim of this research was to understand the influence of the lyophilization cooling profile (including annealing) on the resulting cake structure and reconstitution time. Three protein formulations (BSA 50 mg/ml, BSA 200 mg/ml and IgG1 40 mg/ml, all in 7% w/v sucrose) were investigated after cooling at either 0.5 °C/min, or quench cooling with liquid nitrogen with/without annealing. Significantly longer reconstitution times were observed for the lower protein concentration formulations following quench cool. Porosity measurements found concomitant increases in the surface area of the porous cake structure but a reduction in total pore volume. We propose that slow reconstitution results from either closed pores or small pores impeding the penetration of water into the lyophilized cake

Exploring personalized structural connectomics for moderate to severe traumatic brain injury

AbstractGraph theoretical analysis of the structural connectome has been employed successfully to characterize brain network alterations in patients with traumatic brain injury (TBI). However, heterogeneity in neuropathology is a well-known issue in the TBI population, such that group comparisons of patients against controls are confounded by within-group variability. Recently, novel single-subject profiling approaches have been developed to capture inter-patient heterogeneity. We present a personalized connectomics approach that examines structural brain alterations in five chronic patients with moderate to severe TBI who underwent anatomical and diffusion magnetic resonance imaging. We generated individualized profiles of lesion characteristics and network measures (including personalized graph metric GraphMe plots, and nodal and edge-based brain network alterations) and compared them against healthy reference cases (N = 12) to assess brain damage qualitatively and quantitatively at the individual level. Our findings revealed alterations of brain networks with high variability between patients. With validation and comparison to stratified, normative healthy control comparison cohorts, this approach could be used by clinicians to formulate a neuroscience-guided integrative rehabilitation program for TBI patients, and for designing personalized rehabilitation protocols based on their unique lesion load and connectome

Genomic and transcriptomic variation defines the chromosome-scale assembly of Haemonchus contortus, a model gastrointestinal worm

International audienceHaemonchus contortus is a globally distributed and economically important gastrointestinal pathogen of small ruminants and has become a key nematode model for studying anthelmintic resistance and other parasite-specific traits among a wider group of parasites including major human pathogens. Here, we report using PacBio long-read and OpGen and 10X Genomics long-molecule methods to generate a highly contiguous 283.4 Mbp chromosome-scale genome assembly including a resolved sex chromosome for the MHco3(ISE).N1 isolate. We show a remarkable pattern of conservation of chromosome content with Caenorhabditis elegans, but almost no conservation of gene order. Short and long-read transcriptome sequencing allowed us to define coordinated transcriptional regulation throughout the parasite’s life cycle and refine our understanding of cis- and trans-splicing. Finally, we provide a comprehensive picture of chromosome-wide genetic diversity both within a single isolate and globally. These data provide a high-quality comparison for understanding the evolution and genomics of Caenorhabditis and other nematodes and extend the experimental tractability of this model parasitic nematode in understanding helminth biology, drug discovery and vaccine development, as well as important adaptive traits such as drug resistance

Early ultrasound surveillance of newly-created haemodialysis arteriovenous fistula

IntroductionWe assess if ultrasound surveillance of newly-created arteriovenous fistulas (AVFs) can predict nonmaturation sufficiently reliably to justify randomized controlled trial (RCT) evaluation of ultrasound-directed salvage intervention.MethodsConsenting adults underwent blinded fortnightly ultrasound scanning of their AVF after creation, with scan characteristics that predicted AVF nonmaturation identified by logistic regression modeling.ResultsOf 333 AVFs created, 65.8% matured by 10 weeks. Serial scanning revealed that maturation occurred rapidly, whereas consistently lower fistula flow rates and venous diameters were observed in those that did not mature. Wrist and elbow AVF nonmaturation could be optimally modeled from week 4 ultrasound parameters alone, but with only moderate positive predictive values (PPVs) (wrist, 60.6% [95% confidence interval, CI: 43.9–77.3]; elbow, 66.7% [48.9–84.4]). Moreover, 40 (70.2%) of the 57 AVFs that thrombosed by week 10 had already failed by the week 4 scan, thus limiting the potential of salvage procedures initiated by that scan’s findings to alter overall maturation rates. Modeling of the early ultrasound characteristics could also predict primary patency failure at 6 months; however, that model performed poorly at predicting assisted primary failure (those AVFs that failed despite a salvage attempt), partly because patency of at-risk AVFs was maintained by successful salvage performed without recourse to the early scan data.ConclusionEarly ultrasound surveillance may predict fistula maturation, but is likely, at best, to result in only very modest improvements in fistula patency. Power calculations suggest that an impractically large number of participants (>1700) would be required for formal RCT evaluation