Identification and quantification of chain-pairing variants or mispaired species of asymmetric monovalent bispecific IgG1 monoclonal antibody format using reverse-phase polyphenyl chromatography coupled electrospray ionization mass spectrometry.

Developing a knob-into-hole asymmetric bispecific IgG1 monoclonal antibody (mAb) poses manufacturing challenges due to the expression of chain pairing variants, also called mispaired species, in the desired product. The incorrect pairing of light and heavy chains could result in heterogeneous mispaired species of homodimers, heterodimers, light chain swapping, and low molecular weight species (LMWS). Standard chromatography, capillary electrophoretic, or spectroscopic methods poorly resolve these from the main variants. Here, we report a highly sensitive reverse-phase polyphenyl ultra-high-performance liquid chromatography (RP-UHPLC) method to accurately measure mispaired species of Duet mAb format, an asymmetric IgG1 bispecific mAb, for both process development and quality control analytical tests. Coupled with electrospray ionization mass spectrometry (ESI-MS), it enabled direct online characterization of mispaired species. This single direct assay detected diverse mispaired IgG-like species and LMWS. The method resolved eight disulfide bonds dissociated LMWS and three mispaired LMWS. It also resolved three different types of IgG-like mispaired species, including two homodimers and one heterodimer. The characterization and quantification simultaneously enabled the cell line selection that produces a lesser heterogeneity and lower levels of mispaired species with the desired correctly paired product. The biological activity assessment of samples with increased levels of these species quantified by the method exhibited a linear decline in potency with increasing levels of mispaired species in the desired product. We also demonstrated the utility of the technique for testing in-process intermediate materials to determine and assess downstream purification process capability in removing diverse mispaired IgG-like species and LMWS to a certain level during the downstream purification process. Our investigation demonstrates that adopting this method was vital in developing asymmetric bispecific mAb from the initial stage of cell line development to manufacturing process development. Therefore, this tool could be used in the control strategy to monitor and control mispaired species during manufacturing, thus improving the quality control of the final product.

Comparison of Tetrasodium EDTA 4% with Sodium Citrate 4% as Line-Locking Solutions at 2 Tertiary Hemodialysis Centres.

Background: The patency of central venous catheters (CVCs) in patients undergoing hemodialysis (HD) is maintained by instilling sodium citrate 4% (SC 4%) locking solution. Alteplase, a thrombolytic agent, is administered to restore function if patency is lost. Objective: To compare SC 4% with a new line-locking solution, ethylenediaminetetraacetic acid 4% (EDTA 4%), in terms of CVC patency and alteplase use. Methods: This retrospective chart review included all HD patients who were switched from SC 4% to EDTA 4% locking solution at 2 tertiary HD centres between June and December 2021. Patients were switched to EDTA 4% if they had high usage of alteplase (receiving ≥ 2 doses of alteplase in a 2-week period). For each line-locking agent, HD pump speeds and alteplase use were analyzed over 2 consecutive 12-week periods. Mean serum calcium and ionized calcium values were recorded during each period. A cost analysis was also performed. Results: A total of 37 HD patients were switched to EDTA 4% during the study period. There was no difference in mean HD pump speed between SC 4% and EDTA 4% (307.7 vs 305.1 mL/min, p = 0.48). The number of catheter-use-days on which alteplase was required declined significantly, from 313 days with SC 4% to 94 days with EDTA 4% (p < 0.001), with an overall cost reduction of 34% ($13 183.21). The decrease in alteplase usage was primarily driven by 1 of the 2 sites. A statistically significant decrease in mean ionized calcium at site 2 (from 1.12 to 1.1 mmol/L, p = 0.037) was noted. As well, an intraluminal interaction between EDTA 4% and serum calcium caused 6 cases of low serum calcium. Conclusions: This study showed that use of EDTA 4% as a line-locking agent reduced alteplase usage in the CVCs of HD patients while maintaining adequate pump speed (i.e., ≥ 300 mL/min).

Contexte: La perméabilité des cathéters veineux centraux (CVC) chez les patients hémodialysés (HD) est maintenue en instillant une solution de verrouillage de citrate de sodium à 4 % (CS 4 %). L'alteplase, un agent thrombolytique, est administré pour rétablir la fonction en cas de perte de perméabilité. Objectif: Comparer la solution de CS 4 % et une nouvelle solution de verrouillage, l'acide éthylènediaminetétraacétique 4 % (EDTA 4 %), en termes de perméabilité du CVC et d'utilisation de l'alteplase. Méthodes: Cet examen rétrospectif des dossiers a été réalisé pour tous les patients HD qui sont passés de la solution de verrouillage de CS 4 % à la solution d'EDTA 4 % dans 2 centres d'hémodialyse tertiaires au cours de la période de juin à décembre 2021. Les patients sont passés à l'EDTA 4 % en cas d'utilisation élevée de l'alteplase (≥ 2 doses d'alteplase reçues sur une période de 2 semaines). Pour chaque agent de verrouillage, les vitesses de la pompe d'hémodialyse et l'utilisation de l'alteplase ont été analysées sur 2 périodes consécutives de 12 semaines. Les valeurs moyennes de calcium sérique et de calcium ionisé ont été enregistrées au cours de chaque période. Une analyse des coûts a également été réalisée. Résultats: Au total, 37 patients HD sont passés à l'EDTA 4 % au cours de la période de l'étude. Aucune différence dans la vitesse moyenne de la pompe d'hémodialyse n'a été constatée en cas d'utilisation de la solution de CS 4 % ou d'EDTA 4 % (307,7 c. 305,1 mL/min, p = 0,48). Le nombre de jours d'utilisation du cathéter qui ont nécessité l'utilisation de l'alteplase a diminué de manière significative, passant de 313 jours avec la solution de CS 4 % à 94 jours avec l'EDTA 4 % (p < 0,001); la réduction globale des coûts se montait à 34 % (économies de 13 183,21 $). L'utilisation moins importante de l'alteplase était principalement due à 1 des 2 sites. Une diminution significative du calcium ionisé moyen (1,12 c. 1,1 mmol/L, p = 0,037) a été observée au deuxième site. De plus, une interaction intraluminale entre l'EDTA 4 % et le calcium sérique a provoqué 6 cas d'hypocalcémie. Conclusions: Cette étude a montré que l'utilisation de l'EDTA 4 % comme agent de verrouillage réduisait l'utilisation de l'alteplase dans les CVC des patients HD tout en maintenant une vitesse de pompe adéquate (c'est-à-dire ≥ 300 mL/min).

Parents' and nurses' perceptions and behaviours of family-centred care during periods of busyness.

PURPOSE: Busyness as a construct within modern healthcare is complex and multidimensional. To date, few studies have sought to explore how busyness influences family-centred care. This study explored the influence of busyness on the delivery of family-centred care for nurses and parents. DESIGN AND METHOD: Ethnography was selected as the research design. The study site was a metropolitan tertiary hospital inpatient paediatric unit in Sydney, Australia. Semi-structured interview and non-participant observation techniques were used for data collection. Ten paediatric nurses and 10 parents were interviewed and 40 h of non-participant observations were undertaken. The COREQ was used to report the study. RESULTS: The findings are presented as three key themes: (i) 'Supporting family-centred care' in which participants detail beliefs about the nurse-parent relationships and how despite busyness nurses sought out moments to engage with parents; (ii) 'Being present at the bedside' identified the challenges in optimising safety and how parents adapted their way of being and interacting on the unit; and (iii) 'The emotional cost of busyness' and how this influenced nurse-parent interactions, care delivery and family-centred care. CONCLUSIONS: The ethnography has given shape to social understandings of busyness, the complexities of paediatric nursing and family-centred care. The culture of care changed in moments of busyness and transformed parent and nursing roles, expectations and collaborative care that at time generated internal emotional conflict and tension. PRACTICE IMPLICATIONS: Given the increasing work demands across health systems, new agile ways of working need to ensure maintenance of a family-centred approach. Strategies need to be developed during periods of busyness to better support collaborative connections and the well-being of paediatric nurses and parents. At an organisational level, fostering a positive workplace culture that shares a vision for family-centred care and collaboration is essential. PATIENT OR PUBLIC CONTRIBUTION: Parents of sick children admitted to an acute paediatric inpatient ward were invited to be a participant in a single interview. Parents were aware of the study through ward advertisement and informal discussions with the researchers or senior clinical staff. Engagement with parents was important as healthcare delivery in paediatrics is focused on the delivery of family-centred care. To minimise the risk of child distress and separation anxiety, children were present during the parent interview. Whist children and young people voices were not silenced during the interview process, for this study the parent's voice remained the focus. While important, due to limited resources, parents were not involved in the design analysis or interpretation of the data or in the preparation of this manuscript. DATA SHARING: The data that support the findings of this study are available from the corresponding author upon reasonable request.

2023 EULAR recommendations for the management of fatigue in people with inflammatory rheumatic and musculoskeletal diseases.

OBJECTIVES: Fatigue is prevalent in people with inflammatory rheumatic and musculoskeletal diseases (I-RMDs) and recognised as one of the most challenging symptoms to manage. The existence of multiple factors associated with driving and maintaining fatigue, and the evidence about what improves fatigue has led to a multifaceted approach to its management. However, there are no recommendations for fatigue management in people with I-RMDs. This lack of guidance is challenging for those living with fatigue and health professionals delivering clinical care. Therefore, our aim was to develop EULAR recommendations for the management of fatigue in people with I-RMDs. METHODS: A multidisciplinary taskforce comprising 26 members from 14 European countries was convened, and two systematic reviews were conducted. The taskforce developed the recommendations based on the systematic review of evidence supplemented with taskforce members' experience of fatigue in I-RMDs. RESULTS: Four overarching principles (OAPs) and four recommendations were developed. OAPs include health professionals' awareness that fatigue encompasses multiple biological, psychological and social factors which should inform clinical care. Fatigue should be monitored and assessed, and people with I-RMDs should be offered management options. Recommendations include offering tailored physical activity and/or tailored psychoeducational interventions and/or, if clinically indicated, immunomodulatory treatment initiation or change. Patient-centred fatigue management should consider the individual's needs and preferences, their clinical disease activity, comorbidities and other psychosocial and contextual factors through shared decision-making. CONCLUSIONS: These 2023 EULAR recommendations provide consensus and up-to-date guidance on fatigue management in people with I-RMDs.

Variability in Reporting eGFR at Dialysis Initiation in Canada: A Research Letter.

Background: Estimated glomerular filtration rate (eGFR) at dialysis initiation is increasingly recognized as a key quality indicator (QI) for patients with end-stage kidney disease (ESKD). Specifically, guidelines recommend assessing deferral of dialysis initiation until symptoms arise or if the eGFR is ≤6 mL/min/1.73 m2. Despite the recognition of the importance of this QI, how eGFR at the time of dialysis initiation is defined, collected, and tracked at dialysis centers across Canada remains unknown. Objectives: To identify how provincial renal programs define eGFR at dialysis initiation, to compare practice across Canadian provinces, and to determine if there is a consistent benchmark for deferred dialysis start. Design: Cross-sectional survey distributed to the medical leads of each provincial renal program, administered from July 2021 to November 2021. Quebec was not included given it did not yet participate in Canadian Organ Replacement Register (CORR) data submission. Setting: The survey was designed and distributed by the Canadian Society of Nephrology Quality Improvement & Implementation Science Committee (CSN-QUIS) Indicator Working Group. Methods: The survey asked respondents on how eGFR is defined, collected, reported, and perceived barriers to QI data collection. The National Senior Renal Leaders Forum helped identify the key provincial medical leads to disseminate the survey for completion. Results: Surveys were distributed to the medical leads of the 9 provincial renal programs that participate in CORR. In total, there were 8 responses. Five provinces submit eGFR for all new dialysis starts and 3 provinces only submit this information for chronic patients. There is variation in determining when a patient with acute kidney injury requiring dialysis is classified as a chronic patient. Four provinces use a 30-day trigger, 3 provinces use a 90-day trigger, and the patient's nephrologist makes this determination in 1 province. The creatinine used for the eGFR at dialysis initiation was the value measured on the first dialysis session (ie, day 0) for 5 provinces; the last outpatient clinic creatinine value in 2 provinces, and 1 province did not have a standard definition. Three provinces did not have a benchmark target for eGFR at dialysis initiation, 1 province had a target of <9.5 mL/min/1.73 m2, 3 provinces had a target of <10 mL/min/1.73 m2, 1 province had a target of <15 mL/min/1.73 m2. All 8 responding provincial medical leads support the establishment of a national benchmark for this measure. Limitations: This survey was restricted to provincial medical leads and therefore is unable to determine practice at individual dialysis sites. The survey was not anonymous, so it may be subject to conformity bias. Conclusions: There is wide variability in how eGFR at dialysis initiation is measured and reported across Canada. Additionally, there is no consensus on a benchmark target for an intent-to-defer dialysis strategy. Standardization of target eGFR at dialysis initiation may facilitate national reporting and quality improvement initiatives.

Contexte: Le débit de filtration glomérulaire estimé (DFGe) à l'amorce de la dialyse est de plus en plus reconnu comme un indicateur clé de la qualité (IQ) chez les patients atteints d'insuffisance rénale terminale (IRT). Plus précisément, les lignes directrices recommandent d'évaluer la possibilité de reporter l'initiation de la dialyse jusqu'à l'apparition des symptômes ou l'atteinte d'un DFGe égal ou inférieur à 6 ml/min/1,73 m2. Bien qu'on reconnaisse l'importance de cet IQ, on ignore encore comment le DFGe est défini, mesuré et suivi au moment de l'initiation de la dialyse dans les centres de dialyse canadiens. Objectifs: Déterminer la façon dont les programmes rénaux provinciaux définissent le DFGe à l'initiation de la dialyse, comparer les pratiques en cours dans les différentes provinces canadiennes et déterminer s'il existe une cible de référence commune pour une initiation différée de la dialyse. Conception: Un sondage transversal distribué entre juillet et novembre 2021 aux directeurs médicaux de chaque programme provincial de soins rénaux. Le Québec n'a pas été inclus puisque la province n'a pas encore participé au Registre canadien des insuffisances et des transplantations d'organes (RCITO). Cadre: Le sondage a été conçu et distribué par le Groupe de travail sur les indicateurs du Quality Improvement & Implementation Science Committee de la Société canadienne de néphrologie (CSN-QUIS). Méthodologie: Les répondants au sondage devaient décrire la façon dont le DFGe est défini, mesuré et rapporté, ainsi que les obstacles perçus à la collecte de données sur les IQ. Le sondage a été distribué aux directeurs médicaux provinciaux identifiés par le biais du National Senior Renal leaders Forum. Résultats: Le sondage a été distribué aux directeurs médicaux des neuf programmes provinciaux de soins rénaux participant au RCITO; huit ont répondu. Cinq provinces soumettent le DFGe pour toute nouvelle initiation d'un traitement de dialyse; trois provinces ne soumettent cette information que pour les patients atteints d'insuffisance rénale chronique. Il existe des différences entre les provinces dans la détermination du moment où un patient passe de l'insuffisance rénale aiguë nécessitant une dialyse à l'insuffisance rénale chronique. Quatre provinces utilisent un délai de 30 jours, trois provinces utilisent un délai de 90 jours et dans la dernière province, cette détermination est faite par le néphrologue du patient. Dans cinq des huit provinces sondées, le taux de créatinine utilisé pour établir le DFGe à l'initiation de la dialyse est la valeur mesurée à la première séance de dialyse (au jour 0); deux provinces utilisent la valeur de créatinine mesurée lors de la dernière visite en ambulatoire, et une province n'a pas de définition normalisée. Trois provinces n'ont pas de cible de référence pour le DFGe à l'initiation de la dialyse; cette cible est de moins de 9,5 ml/min/1,73 m2 dans une province, de moins de 10 ml/min/1,73 m2 dans trois provinces, et de moins de 15 ml/min/1,73 m2 dans une province. Les huit responsables médicaux provinciaux ayant répondu au sondage appuient l'établissement d'une valeur de référence nationale pour cette mesure. Limites: Ce sondage n'a été envoyé qu'aux directions médicales provinciales, par conséquent, il ne permet pas de déterminer les pratiques en cours dans chaque site de dialyse. Le sondage n'étant pas anonyme, il pourrait comporter un biais de conformité. Conclusion: Il existe une grande variabilité au Canada dans la façon dont le DFGe est mesuré et rapporté au début de la dialyse. On observe en outre une absence de consensus quant à une cible de référence pour une stratégie d'initiation différée de la dialyse. La normalisation de la valeur cible de DFGe au début de la dialyse pourrait faciliter les initiatives nationales de déclaration et d'amélioration de la qualité.

Barriers and enablers for safe medication administration in adult and neonatal intensive care units mapped to the behaviour change wheel.

BACKGROUND: Intensive care settings have high rates of medication administration errors. Medications are often administered by nurses and midwives using a specified process (the '5 rights'). Understanding where medication errors occur, the contributing factors and how best practice is delivered may assist in developing interventions to improve medication safety. AIMS: To identify medication administration errors and context specific barriers and enablers for best practice in an adult and a neonatal intensive care unit. Secondary aims were to identify intervention functions (through the Behaviour Change Wheel). STUDY DESIGN: A dual methods exploratory descriptive study was conducted (May to June 2021) in a mixed 56-bedded adult intensive care unit and a 6-bedded neonatal intensive care unit in Sydney, Australia. Incident monitoring data were examined. Direct semi-covert observational medication administration audits using the 5 rights (n = 39) were conducted. Brief interviews with patients, parents and nurses were conducted. Data were mapped to the Behaviour Change Wheel. RESULTS: No medication administration incidents were recorded. Audits (n = 3) for the neonatal intensive care unit revealed no areas for improvement. Adult intensive care unit nurses (n = 36) performed checks for the right medication 35 times (97%) and patient identity 25 times (69%). Sixteen administrations (44%) were interrupted. Four themes were synthesized from the interview data: Trust in the nursing profession; Availability of policies and procedures; Adherence to the '5 rights' and departmental culture; and Adequate staffing. The interventional functions most likely to bring about behaviour change were environmental restructuring, enablement, restrictions, education, persuasion and modelling. CONCLUSIONS: This study reveals insights about the medication administration practices of nurses in intensive care. Although there were areas for improvement there was widespread awareness among nurses regarding their responsibilities to safely administer medications. Interview data indicated high levels of trust among patients and parents in the nurses. RELEVANCE TO CLINICAL PRACTICE: This novel study indicated that nurses in intensive care are aware of their responsibilities to safely administer medications. Mapping of contextual data to the Behaviour Change Wheel resulted in the identification of Intervention functions most likely to change medication administration practices in the adult intensive care setting that is environmental restructuring, enablement, restrictions, education, persuasion and modelling.

Interplay of heavy chain introns influences efficient transcript splicing and affects product quality of recombinant biotherapeutic antibodies from CHO cells.

Introns are included in genes encoding therapeutic proteins for their well-documented function of boosting expression. However, mis-splicing of introns in recombinant immunoglobulin (IgG) heavy chain (HC) transcripts can produce amino acid sequence product variants. These variants can affect product quality; therefore, purification process optimization may be needed to remove them, or if they cannot be removed, then in-depth characterization must be carried out to understand their effects on biological activity. In this study, HC transgene engineering approaches were investigated and were successful in significantly reducing the previously identified IgG HC splice variants to <0.5%. Subsequently, a comprehensive evaluation was conducted to understand the influence of the different introns in the HC genes on the expression of recombinant biotherapeutic antibodies. The data revealed an unexpected cooperation between specific introns for efficient splicing, where intron retention led to significant reductions in IgG expression of up to 75% for some intron combinations. Furthermore, it was shown that HC introns could be fully removed without significantly affecting productivity. This work paves the way for future biotherapeutic antibody transgene design with regard to inclusion of HC introns. By removing unnecessary introns, transgene mRNA transcript will no longer be mis-spliced, thereby eliminating HC splice variants and improving antibody product quality.

CHO synthetic promoters improve expression and product quality of biotherapeutic proteins.

When expressing complex biotherapeutic proteins, traditional expression plasmids and methods may not always yield sufficient levels of high-quality product. High-strength viral promoters commonly used for recombinant protein (rProtein) production in mammalian cells allow for maximal expression, but provide limited scope to alter their transcription dynamics. However, synthetic promoters designed to provide tunable transcriptional activity offer a plasmid engineering approach to more precisely regulate product quality, yield or to reduce product related contaminants. We substituted the viral promoter CMV with synthetic promoters that offer different transcriptional activities to express our gene of interest in Chinese hamster ovary (CHO) cells. Stable pools were established and the benefits of regulating transgene transcription on the quality of biotherapeutics were examined in stable pool fed-batch overgrow experiments. Specific control of gene expression of the heavy chain (HC):light chain (LC) of a Fab, and the ratio between the two HCs in a Duet mAb reduced levels of aberrant protein contaminants; and the controlled expression of the helper gene XBP-1s improved expression of a difficult-to-express mAb. This synthetic promoter technology benefits applications that require custom activity. Our work highlights the advantages of employing synthetic promoters for production of more complex rProteins.

Bruch's Membrane: A Key Consideration with Complement-Based Therapies for Age-Related Macular Degeneration.

The complement system is crucial for immune surveillance, providing the body's first line of defence against pathogens. However, an imbalance in its regulators can lead to inappropriate overactivation, resulting in diseases such as age-related macular degeneration (AMD), a leading cause of irreversible blindness globally affecting around 200 million people. Complement activation in AMD is believed to begin in the choriocapillaris, but it also plays a critical role in the subretinal and retinal pigment epithelium (RPE) spaces. Bruch's membrane (BrM) acts as a barrier between the retina/RPE and choroid, hindering complement protein diffusion. This impediment increases with age and AMD, leading to compartmentalisation of complement activation. In this review, we comprehensively examine the structure and function of BrM, including its age-related changes visible through in vivo imaging, and the consequences of complement dysfunction on AMD pathogenesis. We also explore the potential and limitations of various delivery routes (systemic, intravitreal, subretinal, and suprachoroidal) for safe and effective delivery of conventional and gene therapy-based complement inhibitors to treat AMD. Further research is needed to understand the diffusion of complement proteins across BrM and optimise therapeutic delivery to the retina.

Pre-eclampsia is associated with complement pathway activation in the maternal and fetal circulation, and placental tissue.

OBJECTIVES: Pre-eclampsia (PE) is a leading cause of obstetric morbidity, with no definitive therapy other than delivery. We aimed to compare complement markers in maternal and fetal circulation, and placental tissue, between women with PE and healthy pregnant controls. STUDY DESIGN: Maternal and umbilical cord blood was tested for iC3b, C3, C4, properdin, Ba and C5b-9, and placental tissue for C3d, C4d, C9 and C1q, from women with PE (n = 34) and healthy pregnant controls (n = 33). Maternal properdin and Ba tests were repeated in a separate validation cohort (PE n = 35; healthy pregnant controls n = 35). MAIN OUTCOME MEASURES: Complement concentrations in maternal and umbilical cord blood, and placental immunohistochemical complement deposition. RESULTS: Women with PE had significantly lower concentrations of properdin (mean: 4828 vs 6877 ng/ml, p < 0.001) and C4 (mean: 0.20 vs 0.31 g/l, p < 0.001), and higher Ba (median: 150 vs 113 ng/ml, p = 0.012), compared to controls. After controlling for gestational age at blood draw, average properdin concentration was 1945 ng/ml lower in PE vs controls (95 % CI: 1487-2402, p < 0.001). Of the cord blood markers assessed, only Ba differed significantly between PE and controls (median: 337 vs 233 ng/ml, p = 0.004). C4d staining of the syncytiotrophoblast membrane was increased in PE vs controls (median immunoreactivity score 3 vs 0, p < 0.001). Maternal properdin and C4 were significantly negatively correlated with placental C4d staining. CONCLUSIONS: Our data confirm excessive placental complement deposition associated with significant concurrent changes in maternal and fetal circulating complement biomarkers in PE. Inhibition of complement activation is a potential therapeutic target.

The case for complement component 5 as a target in neurodegenerative disease.

INTRODUCTION: Complement-based drug discovery is undergoing a renaissance, empowered by new advances in structural biology, complement biology and drug development. Certain components of the complement pathway, particularly C1q and C3, have been extensively studied in the context of neurodegenerative disease, and established as key therapeutic targets. C5 also has huge therapeutic potential in this arena, with its druggability clearly demonstrated by the success of C5-inhibitor eculizumab. AREAS COVERED: We will discuss the evidence supporting C5 as a target in neurodegenerative disease, along with the current progress in developing different classes of C5 inhibitors and the gaps in knowledge that will help progress in the field. EXPERT OPINION: Validation of C5 as a therapeutic target for neurodegenerative disease would represent a major step forward for complement therapeutics research and has the potential to furnish disease-modifying drugs for millions of patients suffering worldwide. Key hurdles that need to be overcome for this to be achieved are understanding how C5a and C5b should be targeted to bring therapeutic benefit and demonstrating the ability to target C5 without creating vulnerability to infection in patients. This requires greater biological elucidation of its precise role in disease pathogenesis, supported by better chemical/biological tools.

Magnesium and Fracture Risk in the General Population and Patients Receiving Dialysis: A Narrative Review.

Purpose of Review: Magnesium is an essential mineral for bone metabolism, but little is known about how magnesium intake alters fracture risk. We conducted a narrative review to better understand how magnesium intake, through supplementation, diet, or altering the concentration of dialysate magnesium, affects mineral bone disease and the risk of fracture in individuals across the spectrum of kidney disease. Sources of Information: Peer-reviewed clinical trials and observational studies. Methods: We searched for relevant articles in MEDLINE and EMBASE databases. The methodologic quality of clinical trials was assessed using a modified version of the Downs and Black criteria checklist. Key Findings: The role of magnesium intake in fracture prevention is unclear in both the general population and in patients receiving maintenance dialysis. In those with normal kidney function, 2 meta-analyses showed higher bone mineral density in those with higher dietary magnesium, whereas 1 systematic review showed no effect on fracture risk. In patients receiving maintenance hemodialysis or peritoneal dialysis, a higher concentration of dialysate magnesium is associated with a lower concentration of parathyroid hormone, but little is known about other bone-related outcomes. In 2 observational studies of patients receiving hemodialysis, a higher concentration of serum magnesium was associated with a lower risk of hip fracture. Limitations: This narrative review included only articles written in English. Observed effects of magnesium intake in the general population may not be applicable to those with chronic kidney disease particularly in those receiving dialysis.

Justification: Le magnésium est un minéral essentiel pour le métabolisme osseux, mais on en sait peu sur la façon dont un apport en magnésium modifie le risque de fracture. Nous avons procédé à un examen narratif afin de mieux comprendre comment les maladies liées à la densité minérale osseuse et le risque de fracture sont affectés par un apport en magnésium (supplémentation, régime alimentaire ou modification de la concentration de dialysat de magnésium) chez les personnes atteintes d'insuffisance rénale. Sources: Essais cliniques et études observationnelles examinés par des pairs. Méthodologie: Nous avons répertorié les articles pertinents dans les bases de données MEDLINE et EMBASE. Une version modifiée des critères de contrôle de la qualité des études de Downs et Black a servi à évaluer la qualité méthodologique des essais cliniques retenus. Principaux résultats: Le rôle d'un apport en magnésium dans la prévention des fractures n'est pas clair, tant dans la population générale que chez les patients sous dialyse d'entretien. Chez les personnes ayant une fonction rénale normale, deux méta-analyses ont montré que les personnes dont le régime alimentaire est riche en magnésium présentent une densité minérale osseuse plus élevée; alors qu'une revue systématique n'a montré aucun effet sur le risque de fracture. Chez les patients sous hémodialyse d'entretien ou dialyse péritonéale, une concentration plus élevée de dialysat de magnésium est associée à une plus faible concentration d'hormone parathyroïdienne, mais on en sait peu sur les autres effets liés aux os. Dans deux études observationnelles portant sur des patients sous hémodialyse, une concentration plus élevée de magnésium sérique a été associée à un risque plus faible de fracture de la hanche. Limites: Cet examen narratif ne comprend que des articles rédigés en anglais. Il est possible que les effets d'un apport en magnésium observés dans la population générale ne puissent s'appliquer aux personnes atteintes d'une néphropathie chronique, en particulier aux personnes sous dialyse.

Gene targeting as a therapeutic avenue in diseases mediated by the complement alternative pathway.

The complement alternative pathway (AP) is implicated in numerous diseases affecting many organs, ranging from the rare hematological disease paroxysmal nocturnal hemoglobinuria (PNH), to the common blinding disease age-related macular degeneration (AMD). Critically, the AP amplifies any activating trigger driving a downstream inflammatory response; thus, components of the pathway have become targets for drugs of varying modality. Recent validation from clinical trials using drug modalities such as inhibitory antibodies has paved the path for gene targeting of the AP or downstream effectors. Gene targeting in the complement field currently focuses on supplementation or suppression of complement regulators in AMD and PNH, largely because the eye and liver are highly amenable to drug delivery through local (eye) or systemic (liver) routes. Targeting the liver could facilitate treatment of numerous diseases as this organ generates most of the systemic complement pool. This review explains key concepts of RNA and DNA targeting and discusses assets in clinical development for the treatment of diseases driven by the alternative pathway, including the RNA-targeting therapeutics ALN-CC5, ARO-C3, and IONIS-FB-LRX, and the gene therapies GT005 and HMR59. These therapies are but the spearhead of potential drug candidates that might revolutionize the field in coming years.

Alternative pathway dysregulation in tissues drives sustained complement activation and predicts outcome across the disease course in COVID-19.

Complement, a critical defence against pathogens, has been implicated as a driver of pathology in COVID-19. Complement activation products are detected in plasma and tissues and complement blockade is considered for therapy. To delineate roles of complement in immunopathogenesis, we undertook the largest comprehensive study of complement in COVID-19 to date, comprehensive profiling of 16 complement biomarkers, including key components, regulators and activation products, in 966 plasma samples from 682 hospitalized COVID-19 patients collected across the hospitalization period as part of the UK ISARIC4C (International Acute Respiratory and Emerging Infection Consortium) study. Unsupervised clustering of complement biomarkers mapped to disease severity and supervised machine learning identified marker sets in early samples that predicted peak severity. Compared to healthy controls, complement proteins and activation products (Ba, iC3b, terminal complement complex) were significantly altered in COVID-19 admission samples in all severity groups. Elevated alternative pathway activation markers (Ba and iC3b) and decreased alternative pathway regulator (properdin) in admission samples were associated with more severe disease and risk of death. Levels of most complement biomarkers were reduced in severe disease, consistent with consumption and tissue deposition. Latent class mixed modelling and cumulative incidence analysis identified the trajectory of increase of Ba to be a strong predictor of peak COVID-19 disease severity and death. The data demonstrate that early-onset, uncontrolled activation of complement, driven by sustained and progressive amplification through the alternative pathway amplification loop is a ubiquitous feature of COVID-19, further exacerbated in severe disease. These findings provide novel insights into COVID-19 immunopathogenesis and inform strategies for therapeutic intervention.

An assessment of prevalence of Type 1 CFI rare variants in European AMD, and why lack of broader genetic data hinders development of new treatments and healthcare access.

PURPOSE: Advanced age-related macular degeneration (AAMD) risk is associated with rare complement Factor I (FI) genetic variants associated with low FI protein levels (termed 'Type 1'), but it is unclear how variant prevalences differ between AMD patients from different ethnicities. METHODS: Collective prevalence of Type 1 CFI rare variant genotypes were examined in four European AAMD datasets. Collective minor allele frequencies (MAFs) were sourced from the natural history study SCOPE, the UK Biobank, the International AMD Genomics Consortium (IAMDGC), and the Finnish Biobank Cooperative (FINBB), and compared to paired control MAFs or background population prevalence rates from the Genome Aggregation Database (gnomAD). Due to a lack of available genetic data in non-European AAMD, power calculations were undertaken to estimate the AAMD population sizes required to identify statistically significant association between Type 1 CFI rare variants and disease risk in different ethnicities, using gnomAD populations as controls. RESULTS: Type 1 CFI rare variants were enriched in all European AAMD cohorts, with odds ratios (ORs) ranging between 3.1 and 7.8, and a greater enrichment was observed in dry AMD from FINBB (OR 8.9, 95% CI 1.49-53.31). The lack of available non-European AAMD datasets prevented us exploring this relationship more globally, however a statistical association may be detectable by future sequencing studies that sample approximately 2,000 AAMD individuals from Ashkenazi Jewish and Latino/Admixed American ethnicities. CONCLUSIONS: The relationship between Type 1 CFI rare variants increasing odds of AAMD are well established in Europeans, however the lack of broader genetic data in AAMD has adverse implications for clinical development and future commercialisation strategies of targeted FI therapies in AAMD. These findings emphasise the importance of generating more diverse genetic data in AAMD to improve equity of access to new treatments and address the bias in health care.

Monoclonal Antibody Sequence Variants Disguised as Fragments: Identification, Characterization, and Their Removal by Purification Process Optimization.

During early stage development of a therapeutic IgG1 monoclonal antibody, high levels of low molecular weight (LMW) peaks were observed by high performance size-exclusion chromatography and capillary electrophoresis. Further characterization of the LMW peak enriched HPSEC fractions using reversed phase liquid chromatography coupled to mass spectrometry showed these LMW species were 47 kDa and 50 kDa in size. However, the measured masses could not be matched to any fragments resulting from peptide bond hydrolysis. To identify these unknown LMW species, molecular characterization methods were employed, including high-throughput sequencing of RNA. Transcriptomic analysis revealed the LMW species were generated by mis-splicing events in the heavy chain transcript, which produced truncated heavy chain products that assembled with the light chain to mimic the appearance of fragments identified by routine purity assays. In an effort to improve product quality, an optimized purification process was developed. Characterization of the process intermediates confirmed removal of both LMW species by the optimized process. Our study demonstrates that deep-dive analytical characterization of biotherapeutics is critical to ensure product quality and inform process development. Transcriptomic analysis tools can help identify the cause of unknown species, and plays a key role in product and process characterization.

Visualization for epidemiological modelling: challenges, solutions, reflections and recommendations.

We report on an ongoing collaboration between epidemiological modellers and visualization researchers by documenting and reflecting upon knowledge constructs-a series of ideas, approaches and methods taken from existing visualization research and practice-deployed and developed to support modelling of the COVID-19 pandemic. Structured independent commentary on these efforts is synthesized through iterative reflection to develop: evidence of the effectiveness and value of visualization in this context; open problems upon which the research communities may focus; guidance for future activity of this type and recommendations to safeguard the achievements and promote, advance, secure and prepare for future collaborations of this kind. In describing and comparing a series of related projects that were undertaken in unprecedented conditions, our hope is that this unique report, and its rich interactive supplementary materials, will guide the scientific community in embracing visualization in its observation, analysis and modelling of data as well as in disseminating findings. Equally we hope to encourage the visualization community to engage with impactful science in addressing its emerging data challenges. If we are successful, this showcase of activity may stimulate mutually beneficial engagement between communities with complementary expertise to address problems of significance in epidemiology and beyond. See https://ramp-vis.github.io/RAMPVIS-PhilTransA-Supplement/. This article is part of the theme issue 'Technical challenges of modelling real-life epidemics and examples of overcoming these'.