Antibody glycan quality predicted from CHO cell culture media markers and machine learning.

N-glycosylation can have a profound effect on the quality of mAb therapeutics. In biomanufacturing, one of the ways to influence N-glycosylation patterns is by altering the media used to grow mAb cell expression systems. Here, we explore the potential of machine learning (ML) to forecast the abundances of N-glycan types based on variables related to the growth media. The ML models exploit a dataset consisting of detailed glycomic characterisation of Anti-HER fed-batch bioreactor cell cultures measured daily under 12 different culture conditions, such as changes in levels of dissolved oxygen, pH, temperature, and the use of two different commercially available media. By performing spent media quantitation and subsequent calculation of pseudo cell consumption rates (termed media markers) as inputs to the ML model, we were able to demonstrate a small subset of media markers (18 selected out of 167 mass spectrometry peaks) in a Chinese Hamster Ovary (CHO) cell cultures are important to model N-glycan relative abundances (Regression - correlations between 0.80-0.92; Classification - AUC between 75.0-97.2). The performances suggest the ML models can infer N-glycan critical quality attributes from extracellular media as a proxy. Given its accuracy, we envisage its potential applications in biomaufactucuring, especially in areas of process development, downstream and upstream bioprocessing.

Biomass specific perfusion rate as a control lever for the continuous manufacturing of biosimilar monoclonal antibodies from CHO cell cultures.

Continuous manufacturing enables high volumetric productivities of biologics such as monoclonal antibodies. However, it is challenging to maintain both high viable cell densities and productivities at the same time for long culture durations. One of the key controls in a perfusion process is the perfusion rate which determines the nutrient availability and potentially controls the cell metabolism. Cell Specific Perfusion Rate (CSPR) is a feed rate proportional to the viable cell density while Biomass Specific Perfusion Rate (BSPR) is a feed rate proportional to the biomass (cell volume multiply by cell density). In this study, perfusion cultures were run at three BSPRs in the production phase. Low BSPR favored a growth arresting state that led to gradual increase in cell volume, which in turn led to an increase in net perfusion rate proportional to the increase in cell volume. Consequently, at low BSPR, while the cell viability and cell density decreased, high specific productivity of 55 pg per cell per day was achieved. In contrast, the specific productivity was lower in bioreactors operating at a high BSPR. The ability to modulate the cell metabolism by using BSPR was confirmed when the specific productivity increased after lowering the BSPR in one of the bioreactors that was initially operating at a high BSPR. This study demonstrated that BSPR significantly influenced cell growth, metabolism, and productivity in cultures with variable cell volumes.

Examining the acceptability and feasibility of the Compassionate Mindful Resilience (CMR) programme in adults living with chronic kidney disease: the COSMIC study findings.

BACKGROUND: Individuals with chronic kidney disease experience difficult physical and psychological symptoms, that impact quality of life, and are at increased risk of anxiety and depression. Access to specialist psychological support is limited. This study aimed to support a new service development project, in collaboration with Kidney Care UK, to implement the Compassionate Mindful Resilience (CMR) programme, developed by MindfulnessUK, which provides accessible mindfulness techniques and practices to enhance compassion and resilience, and explore its feasibility for people living with stage 4 or 5 kidney disease and transplant. METHODS: A multi-method feasibility design was utilised. Participants over 18 years, from the UK, with stage 4 or 5 kidney disease or post-transplant, and who were not currently undergoing psychotherapy, were recruited to the four-week CMR programme. Data was collected at baseline, post-intervention and three-months post to measure anxiety, depression, self-compassion, mental wellbeing, resilience, and mindfulness. The acceptability of the intervention for a kidney disease population was explored through qualitative interviews with participants, and the Mindfulness Teacher. RESULTS: In total, 75 participants were recruited to the study, with 65 completing the CMR programme. The majority were female (66.2%) and post-transplant (63.1%). Analysis of completed outcome measures at baseline and post-intervention timepoints (n = 61), and three-months post intervention (n = 45) revealed significant improvements in participant's levels of anxiety (p < .001) and depression (p < .001), self-compassion (p = .005), mental wellbeing (p < .001), resilience (p.001), and mindfulness (p < .001). Thematic analysis of interviews with participants (n = 19) and Mindfulness Teacher (n = 1) generated three themes (and nine-subthemes); experiences of the CMR programme that facilitated subjective benefit, participants lived and shared experiences, and practicalities of programme participation. All participants interviewed reported that they found programme participation to be beneficial. CONCLUSION: The findings suggest that the CMR programme has the potential to improve psychological outcomes among people with chronic kidney disease. Future randomized controlled trials are required to further test its effectiveness.

Experiences of People with Kidney Disease Following the Implementation of the Compassionate Mindful Resilience Programme: Qualitative Findings from the COSMIC Study.

BACKGROUND: Kidney disease is a progressive, debilitating condition. Patients experience challenging physical and psychological symptoms and are at increased risk of anxiety, depression, and poor mental wellbeing. Access to specialist psychological or social support is limited, with inadequate provision of psychosocial support available across UK renal units. The COSMIC study (examining the acceptability and feasibility of the Compassionate Mindful Resilience programme for adult patients with chronic kidney disease) aimed to support a new service development project, in partnership with Kidney Care UK, by implementing the Compassionate Mindful Resilience (CMR) programme, developed by MindfulnessUK, and explore its feasibility for patients with stage 4 or 5 kidney disease and kidney transplant recipients. This paper reports on the qualitative exploratory work which examined the experiences of study participants, their adherence to practice, and the acceptability of the intervention. METHOD: Participants (n = 19) took part in semi-structured interviews, which were transcribed, coded, and thematically analysed. RESULTS: Three themes (and nine subthemes) were reported: experiences of the CMR programme that facilitated subjective benefit, participants' lived and shared experiences, and the practicalities of CMR programme participation. All participants reported that they found taking part in the CMR programme to be a beneficial experience. CONCLUSION: The CMR programme was found to be an acceptable intervention for people living with kidney disease and provided tools and techniques that support the mental health and wellbeing of this patient group. Further qualitative exploration into participant experience should be integrated within future trials of this intervention.

Optimizing effector functions of monoclonal antibodies via tailored N-glycan engineering using a dual landing pad CHO targeted integration platform.

Monoclonal antibodies (mAbs) eliminate cancer cells via various effector mechanisms including antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), which are influenced by the N-glycan structures on the Fc region of mAbs. Manipulating these glycan structures on mAbs allows for optimization of therapeutic benefits associated with effector functions. Traditional approaches such as gene deletion or overexpression often lead to only all-or-nothing changes in gene expression and fail to modulate the expression of multiple genes at defined ratios and levels. In this work, we have developed a CHO cell engineering platform enabling modulation of multiple gene expression to tailor the N-glycan profiles of mAbs for enhanced effector functions. Our platform involves a CHO targeted integration platform with two independent landing pads, allowing expression of multiple genes at two pre-determined genomic sites. By combining with internal ribosome entry site (IRES)-based polycistronic vectors, we simultaneously modulated the expression of α-mannosidase II (MANII) and chimeric ß-1,4-N-acetylglucosaminyl-transferase III (cGNTIII) genes in CHO cells. This strategy enabled the production of mAbs carrying N-glycans with various levels of bisecting and non-fucosylated structures. Importantly, these engineered mAbs exhibited different degrees of effector cell activation and CDC, facilitating the identification of mAbs with optimal effector functions. This platform was demonstrated as a powerful tool for producing antibody therapeutics with tailored effector functions via precise engineering of N-glycan profiles. It holds promise for advancing the field of metabolic engineering in mammalian cells.

Understanding and controlling the molecular mechanisms of protein aggregation in mAb therapeutics.

In antibody development and manufacturing, protein aggregation is a common challenge that can lead to serious efficacy and safety issues. To mitigate this problem, it is important to investigate its molecular origins. This review discusses (1) our current molecular understanding and theoretical models of antibody aggregation, (2) how various stress conditions related to antibody upstream and downstream bioprocesses can trigger aggregation, and (3) current mitigation strategies employed towards inhibiting aggregation. We discuss the relevance of the aggregation phenomenon in the context of novel antibody modalities and highlight how in silico approaches can be exploited to mitigate it.

Insight into muscle stem cell regeneration and mechanobiology.

Stem cells possess the unique ability to differentiate into specialized cell types. These specialized cell types can be used for regenerative medicine purposes such as cell therapy. Myosatellite cells, also known as skeletal muscle stem cells (MuSCs), play important roles in the growth, repair, and regeneration of skeletal muscle tissues. However, despite its therapeutic potential, the successful differentiation, proliferation, and expansion processes of MuSCs remain a significant challenge due to a variety of factors. For example, the growth and differentiation of MuSCs can be greatly influenced by actively replicating the MuSCs microenvironment (known as the niche) using mechanical forces. However, the molecular role of mechanobiology in MuSC growth, proliferation, and differentiation for regenerative medicine is still poorly understood. In this present review, we comprehensively summarize, compare, and critically analyze how different mechanical cues shape stem cell growth, proliferation, differentiation, and their potential role in disease development (Fig. 1). The insights developed from the mechanobiology of stem cells will also contribute to how these applications can be used for regenerative purposes using MuSCs.

Enhancing pharmacokinetic and pharmacodynamic properties of recombinant therapeutic proteins by manipulation of sialic acid content.

The circulatory half-life of recombinant therapeutic proteins is an important pharmacokinetic attribute because it determines the dosing frequency of these drugs, translating directly to treatment cost. Thus, recombinant therapeutic glycoproteins such as monoclonal antibodies have been chemically modified by various means to enhance their circulatory half-life. One approach is to manipulate the N-glycan composition of these agents. Among the many glycan constituents, sialic acid (specifically, N-acetylneuraminic acid) plays a critical role in extending circulatory half-life by masking the terminal galactose that would otherwise be recognised by the hepatic asialoglycoprotein receptor (ASGPR), resulting in clearance of the biotherapeutic from the circulation. This review aims to provide an illustrative overview of various strategies to enhance the pharmacokinetic/pharmacodynamic properties of recombinant therapeutic proteins through manipulation of their sialic acid content.

Computerization of the Work of General Practitioners: Mixed Methods Survey of Final-Year Medical Students in Ireland.

BACKGROUND: The potential for digital health technologies, including machine learning (ML)-enabled tools, to disrupt the medical profession is the subject of ongoing debate within biomedical informatics. OBJECTIVE: We aimed to describe the opinions of final-year medical students in Ireland regarding the potential of future technology to replace or work alongside general practitioners (GPs) in performing key tasks. METHODS: Between March 2019 and April 2020, using a convenience sample, we conducted a mixed methods paper-based survey of final-year medical students. The survey was administered at 4 out of 7 medical schools in Ireland across each of the 4 provinces in the country. Quantitative data were analyzed using descriptive statistics and nonparametric tests. We used thematic content analysis to investigate free-text responses. RESULTS: In total, 43.1% (252/585) of the final-year students at 3 medical schools responded, and data collection at 1 medical school was terminated due to disruptions associated with the COVID-19 pandemic. With regard to forecasting the potential impact of artificial intelligence (AI)/ML on primary care 25 years from now, around half (127/246, 51.6%) of all surveyed students believed the work of GPs will change minimally or not at all. Notably, students who did not intend to enter primary care predicted that AI/ML will have a great impact on the work of GPs. CONCLUSIONS: We caution that without a firm curricular foundation on advances in AI/ML, students may rely on extreme perspectives involving self-preserving optimism biases that demote the impact of advances in technology on primary care on the one hand and technohype on the other. Ultimately, these biases may lead to negative consequences in health care. Improvements in medical education could help prepare tomorrow's doctors to optimize and lead the ethical and evidence-based implementation of AI/ML-enabled tools in medicine for enhancing the care of tomorrow's patients.

The renal-bone axis in older people living with HIV on stable antiretroviral therapy: A sub-analysis of the GS-US-104-0423 study.

BACKGROUND: Data on low bone mineral density (BMD) in people living with HIV (PLWH) are mainly derived from younger adults; little is known about how antiretroviral therapy (ART) and alterations in the renal-bone axis relate to BMD in older PLWH. METHODS: Cross-sectional study of men > 50 years and post-menopausal women with HIV. Antiretroviral therapy exposure was stratified into four groups based on use of tenofovir disoproxil fumarate (TDF) and protease inhibitors (PI): non-TDF/non-PI, non-TDF/PI, TDF/non-PI, and TDF/PI. Bone mineral density was measured by dual X-ray absorptiometry (DXA). Bone turnover/regulatory markers and renal tubular function were analysed in stored plasma and urine samples. The association of ART exposure and bone/renal biomarkers on BMD was explored using logistic regression models. RESULTS: 247 individuals (median [IQR] age 57 [53, 65] years; 47% female; 13% of Black ethnicity; CD4 count 643 [473, 811] cells/mm3; and 98% with HIV RNA < 200 copies/mL) were included. Bone turnover and renal tubular function differed significantly by ART exposure. In analyses adjusted for demographic and traditional renal/bone risk factors, exposure to TDF and PI was associated with a fourfold greater risk of low BMD at the femoral neck and exposure to TDF and/or PI with a threefold greater risk of low BMD at the lumbar spine. The relationship between ART and low BMD was not altered by further adjustment for bone turnover or renal tubular function markers. CONCLUSIONS: The associations between low BMD and ART exposure (TDF vs. non-TDF and boosted vs. unboosted third agents) were minimally affected by adjustments for bone and kidney biomarkers.

An Integrative Glycomic Approach for Quantitative Meat Species Profiling.

It is estimated that food fraud, where meat from different species is deceitfully labelled or contaminated, has cost the global food industry around USD 6.2 to USD 40 billion annually. To overcome this problem, novel and robust quantitative methods are needed to accurately characterise and profile meat samples. In this study, we use a glycomic approach for the profiling of meat from different species. This involves an O-glycan analysis using LC-MS qTOF, and an N-glycan analysis using a high-resolution non-targeted ultra-performance liquid chromatography-fluorescence-mass spectrometry (UPLC-FLR-MS) on chicken, pork, and beef meat samples. Our integrated glycomic approach reveals the distinct glycan profile of chicken, pork, and beef samples; glycosylation attributes such as fucosylation, sialylation, galactosylation, high mannose, α-galactose, Neu5Gc, and Neu5Ac are significantly different between meat from different species. The multi-attribute data consisting of the abundance of each O-glycan and N-glycan structure allows a clear separation between meat from different species through principal component analysis. Altogether, we have successfully demonstrated the use of a glycomics-based workflow to extract multi-attribute data from O-glycan and N-glycan analysis for meat profiling. This established glycoanalytical methodology could be extended to other high-value biotechnology industries for product authentication.

Examining the Acceptability and Feasibility of the Compassionate Mindful Resilience (CMR) Programme in Adult Patients with Chronic Kidney Disease: The COSMIC Study Protocol.

Kidney disease is often progressive, and patients experience diminished health-related quality of life. In addition, the impact of the coronavirus (COVID-19) pandemic, and its associated restrictions, has brought many additional burdens. It is therefore essential that effective and affordable systems are explored to improve the psychological health of this group that can be delivered safely during the COVID-19 pandemic. The aim of this study is to support a new service development project in partnership with the UK's leading patient support charity Kidney Care UK by implementing the four-session Compassionate Mindful Resilience (CMR) programme, developed by MindfulnessUK, and explore its effectiveness for patients with stage 4 or 5 chronic kidney disease or have received a kidney transplant. The study will utilise a quasi-experimental, pretest/posttest design to measure the effect of the CMR programme on anxiety, depression, self-compassion, the ability to be mindful, wellbeing, and resilience, using pre- and posttests, alongside a qualitative exploration to explore factors influencing the feasibility, acceptability, and suitability of the intervention, with patients (and the Mindfulness Teacher) and their commitment to practice. Outcomes from this study will include an evidence-based mindfulness and compassion programme for use with people with kidney disease, which is likely to have applicability across other chronic diseases.

Evaluation of a Programme of Online Arts Activities for Patients with Kidney Disease during the COVID-19 Pandemic.

Patients living with end-stage kidney disease (ESKD) have been seriously impacted by the COVID-19 pandemic. As these patients are considered extremely clinically vulnerable, they were advised to 'shield' at home, with limited face-to-face contact and support for the duration of the pandemic. Living with ESKD impacts heavily on patients' mental health and wellbeing, and this extended period of isolation and loneliness is likely to have a further negative effect on patients' mental wellbeing. The Renal Arts Group (RAG), Queen's University Belfast, aims to improve the quality of life of those living with ESKD and the extended renal community through engagement with the arts. We developed an initiative, funded by the Economic and Social Research Council, and carried out an evaluation. The initiative included a programme of online arts-based activities that built upon the work of RAG and provided mental wellbeing support for patients who faced an extended, lonely period of self-isolation. We worked with experienced arts practitioners to identify appropriate activities and developed five workshops and tutorials that were delivered online. We received positive feedback from participants who found the activities to be enjoyable, beneficial to their mental wellbeing and were interested in undertaking further activities online. We conducted interviews with the arts facilitators and identified three themes for consideration when developing online arts activities for the renal community. Participants reported that the activities benefited their mental wellbeing, were enjoyable and provided an opportunity to meet others with shared interests. The arts facilitators reported experiences around accessibility, audience engagement, impact on health and wellbeing and facilitator experience, that should be considered when developing online arts activities for the renal community. This evaluation will inform future work in this area, and the arts tutorial videos developed as part of this project will remain available online for members of the renal community to access.

Harnessing the potential of machine learning for advancing "Quality by Design" in biomanufacturing.

Ensuring consistent high yields and product quality are key challenges in biomanufacturing. Even minor deviations in critical process parameters (CPPs) such as media and feed compositions can significantly affect product critical quality attributes (CQAs). To identify CPPs and their interdependencies with product yield and CQAs, design of experiments, and multivariate statistical approaches are typically used in industry. Although these models can predict the effect of CPPs on product yield, there is room to improve CQA prediction performance by capturing the complex relationships in high-dimensional data. In this regard, machine learning (ML) approaches offer immense potential in handling non-linear datasets and thus are able to identify new CPPs that could effectively predict the CQAs. ML techniques can also be synergized with mechanistic models as a 'hybrid ML' or 'white box ML' to identify how CPPs affect the product yield and quality mechanistically, thus enabling rational design and control of the bioprocess. In this review, we describe the role of statistical modeling in Quality by Design (QbD) for biomanufacturing, and provide a generic outline on how relevant ML can be used to meaningfully analyze bioprocessing datasets. We then offer our perspectives on how relevant use of ML can accelerate the implementation of systematic QbD within the biopharma 4.0 paradigm.

Glycoinformatics Tools for Comprehensive Characterization of Glycans Enzymatically Released from Proteins.

Glycosylation is important in biology, contributing to both protein conformation and function. Structurally, glycosylation is complex and diverse. This complexity is reflected in the topology, composition, monosaccharide linkages, and isomerism of each oligosaccharide. Glycoanalytics is a discipline that addresses the understanding and characterization of this complexity and its correlation with biology. It includes analytical steps such as sample preparation, instrument measurements, and data analyses. Of these, data analysis has emerged as a critical bottleneck because data collection has increasingly become high-throughput. This has resulted in data-rich workflows that lack rapid and automated data analytics. To address this issue, the field has been developing software for interpretation of quantitative glycomics studies. Here, we describe a protocol using available informatics tools for analysis of data from analysis of released glycans using high-/ultraperformance liquid chromatography (H/UPLC) coupled with mass spectrometry (MS).

Mindfulness as a well-being initiative for future nurses: a survey with undergraduate nursing students.

BACKGROUND: Mindfulness can potentially positively impact well-being and resilience in undergraduate nursing students. The psychological well-being of such students undertaking clinical training is paramount to ensure optimal learning, and to equip them with skills to manage their wellbeing in future clinical practice. The aim of our study was to explore the views of undergraduate nursing students in relation to understanding and engaging with mindfulness, and how mindfulness could best be delivered within their university programme. METHODS: An online survey was administered via a cloud-based student response system to a convenience sample of first year undergraduate nursing students completing a Bachelor of Science (BSc) Honours (Hons) degree in nursing at a University in the United Kingdom. Data were analysed using descriptive statistics and thematic analysis. RESULTS: The survey achieved a response rate of 78% (n = 208). Seventy-nine percent of participants had heard of mindfulness and were interested in taking part in a mindfulness programme. Respondents reported that the ideal delivery of the programme would consist of weekly 45-min, in person group sessions, over a 6-week period. Respondents also indicated that a mobile application could potentially facilitate participation in the programme. Thematic analysis of open-ended comments, and free text, within the survey indicated 4 overarching themes: 1) Perceptions of what mindfulness is; 2) Previous mindfulness practice experiences; 3) Impact of mindfulness in nursing; 4) The need for a future well-being initiative for undergraduate nursing students. CONCLUSIONS: Undergraduate nursing students perceived that a mindfulness programme has the potential to enhance well-being and future clinical practice. This student cohort are familiar with mindfulness and want more integrated within their undergraduate curriculum. Further research is required to examine the effectiveness of a tailored mindfulness intervention for this population that incorporates the use of both face-to-face and mobile delivery.

Exploration of Caregiver Experiences of Conservatively Managed End-Stage Kidney Disease to Inform Development of a Psychosocial Intervention: The ACORN Study Protocol.

BACKGROUND: End-stage kidney disease (ESKD) is an overwhelming illness that impacts not just patients, but also their informal carers. Patients who opt for conservative management rather than dialysis experience difficult symptoms and the psychosocial consequences of their condition. Informal carers of patients who choose conservative management can also experience high levels of psychosocial burden, yet there is little guidance on how best to support informal carers, and no evidence on psychosocial interventions to address unmet needs. AIM: The aim of this study is to explore the experiences and unmet needs of informal carers of patients with ESKD receiving conservative management in order to inform the development of a psychosocial intervention. METHODS: This qualitative study will consist of three stages: (I) semi-structured interviews with informal carers in England and Northern Ireland, (II) focus groups with healthcare professionals and informal carers, and (III) national workshops to refine the components of a psychosocial intervention. DISCUSSION: Informal carers of patients with ESKD who are receiving conservative management experience a high psychosocial burden, but there is limited evidence on how best to provide support, particularly as the patient nears the end of life. To our knowledge this study will be the first to address this gap by exploring the experiences and unmet needs of informal carers, with the aim of informing the development of a psychosocial intervention to support the health and wellbeing of informal carers.

The Experiences of a Complex Arts-Based Intervention for Patients with End-Stage Kidney Disease Whilst Receiving Haemodialysis: A Qualitative Process Evaluation.

The global prevalence and burden of end-stage kidney disease (ESKD) is increasing, partially as a result of an aging population. Patients with ESKD who receive haemodialysis experience a difficult, protracted treatment regimen that can negatively impact mental health and wellbeing. One way of addressing this issue could be through the provision of arts-based interventions during haemodialysis treatment. A process evaluation was conducted as part of a larger feasibility study, to explore experiences and acceptability of an intra-dialytic (during haemodialysis) arts-based intervention. Thirteen patients and nine healthcare professionals were interviewed. The interviews were transcribed verbatim and thematically analysed. Three themes related to the experience of art on dialysis included: the perception of art participation, as patients described initial anxiety around participating in art, which reduced after they experienced the intervention; the benefits of art participation on both patients and healthcare professionals; the benefits including improved self-esteem, motivation, increased social interaction and an overall improved haemodialysis experience; and the acceptability of the arts-based intervention, as the intervention was highly acceptable, even when delivered by a facilitator who was not a professional artist. This study highlights that arts-based interventions could be used to improve the mental health and wellbeing of patients with ESKD receiving haemodialysis.