Development of chemically-defined, animal component-free medium for suspension MDCK cell-based influenza vaccine production

Cell culture-based vaccine production is advantageous over chicken eggs-based production because it does not rely on the availability of eggs and can be performed on a larger scale in a timely manner. Madin-Darby canine kidney (MDCK) cells are favorably used for vaccine production, particularly for influenza vaccines. However, there are several drawbacks to the traditional method of culturing MDCK cells. MDCK cells require undefined supplement for culturing, such as serum or hydrolysates which pose regulatory concerns especially when making pharmaceutical products for human use. Furthermore, MDCK cells grow in adherent culture which requires significant surface area or the use of microcarriers, thus it is more challenging in a large commercial scale process and is more costly compare to a suspension culture process. In this study, we report the development of a chemically-defined (CD) and animal component-free (ACF) medium that was able to adapt an adherent MDCK cell line to grow in suspension without a need of microcarriers. The results show that suspension-adapted MDCK cells in this CD and ACF medium achieved comparable cell growth and influenza production to that of the existing process which uses adherent MDCK cells on microcarriers in an undefined medium. In addition, the cell growth and production of the suspension cultures were consistent over ten passages while maintaining antigenicity of the virus. This newly established chemically-defined, suspension culture process can overcome the drawbacks of the traditional MDCK culture, leading to a more cost-effective, robust, and scalable process for MDCK cell-based vaccine productions

Rapid and flexible scale-down media development and optimization for perfusion culture

Perfusion culture is steadily gaining interest due to advantages such as higher cell densities, increased flexibility, higher volumetric output, decreased product retention times, and more consistent product. Perfusion culture systems are drastically different than fed-batch cultures as they allow bioreactors to continuously run for prolonged periods of time by constantly feeding fresh media while removing spent media. This drastic process difference means that typical fed-batch medium is not optimal for perfusion culture. Instead, perfusion culture media is most optimal when it fits within culture process constraints such as vessel volume exchanges per day. Irvine Scientific has rapidly developed custom media for a specific perfusion culture process that resulted in a significant increase in viable cell density and antibody titer. The custom media was developed in the following steps: (1) identification of initial base media, (2) development and verification of a scale-down perfusion model, (3) optimization of media components, and (4) parsing down extraneous components while maintaining growth and titer. First, a media survey was conducted in batch cultures to determine an initial basal media composition to further refine. From a panel of over 20 different media, top candidate media were selected based on improved viable cell density, viability, titer, and glycan profile over the control medium. Next, a scale-down perfusion model was developed and validated using the control medium. Mini bioreactor tubes were used as culture vessels and daily perfusion was conducted by centrifugation, aspiration, and replenishment with fresh medium. Antibody glycan profiles from the scale-down perfusion model matched profiles from large-scale bioreactor perfusion cultures, thus validating the use of this scale-down model. Although this system is not controlled, the pH was monitored offline and stayed within range. The candidate media were evaluated in the scale-down perfusion model and the top prospects in terms of titer and viable cell density were chosen for further development by changing the composition of amino acids, metals, and other components. Lastly, amino acid concentrations were further optimized while parsing down extraneous components according to spent media analysis from the previous experiment. The top medium resulted in a significant increase in viable cell density and titer compared to the original control medium. This process created a custom medium for perfusion culture that significantly increased viable cell density and antibody titer while maintaining a desired glycan profile. The media development process in scale-down perfusion cultures was rapid and took about 10 weeks. Development in controlled, scale-down bioreactor systems may be more costly and take longer as procedures, protocols, and methods first need to be transferred and verified. This scale-down perfusion model, while manual, is an easy and flexible way to develop perfusion medium with efficient throughput. Currently, the medium is being verified in perfusion bioreactors and can be further improved. In the future, we will investigate increasing the productivity by concentrating the culture medium while decreasing the volume vessel exchanges

Low-cost cell-based production platform for seasonal and pandemic influenza vaccines

Influenza-related illnesses have caused an estimated over million cases of severe illness, and it has about hundred thousands of deaths worldwide annually. Traditionally these vaccines are produced in embryonated chicken eggs. However, in the case of a pandemic outbreak, this egg-based production system may not be quickly enough to meet the surging demand. The efficacy associated with egg-based vaccines are low in recently years. The raising concerns with egg-derived vaccines is resulting in the spurred exploration of alternatives. MDCK cells are becoming as an alternative host to embryonated eggs for influenza virus propagation. Although MDCK cells were considered to be a suitable host for the virus production, their inability to grow in suspension still limits the process of scale-up and their production capability. Please click Additional Files below to see the full abstract

Development of an animal-component free insect medium for the Baculovirus Expression Vector System (BEVS)

Insect cells derived from Spodoptera frugiperda have been widely used with the baculovirus expression vector system (BEVS) for the production of recombinant proteins and adeno-associated viruses (AAVs) due to their ease of culture, scalability in high cell density suspension cultures, and high protein expression levels. Traditionally, insect cells are cultured in an undefined medium containing yeast hydrolysate and cod liver oil, however, there is an increasing push to use chemically defined, animal-component free medium to minimize any potential contaminants and decrease lot-to-lot variability while maintaining high cell growth and production. In this case study, an animal-component free insect medium was developed utilizing Rational Culture Media DesignTM and evaluated with Sf9 cells. Using a traditional formulation as a starting point, the final medium was developed by optimizing multiple nutrient groups in the basal medium, replacing the animal-derived components, and screening several yeast hydrolysate sources. By utilizing multifactor design of experiment software, various nutrient groups were screened including amino acids, vitamins, and metals. The metals group was identified to have the most impact on cell growth and productivity, and therefore concentrations of metal components were further optimized. In addition, the animal-derived components in the starting formulation, cod liver oil and cholesterol, were replaced with animal-component free fatty acids and synthetic cholesterol, respectively. The concentrations of these components were optimized to achieve better growth performance and production while also sustaining formulation stability and streamlining manufacturing processes. Finally, yeast hydrolysate is a well-known, undefined component that is crucial for insect cell growth and productivity. To minimize lot-to-lot variability, the yeast hydrolysate concentration was significantly lowered, and multiple yeast hydrolysate sources and lots were evaluated to determine the highest quality source. As a result, an animal-component free insect medium was developed that had improved growth performance and comparable productivity to a widely used commercially available animal-derived medium

Safety and Feasibility of Research Lumbar Puncture in Huntington’s Disease: The HDClarity Cohort and Bioresource

Background: Biomarkers are needed to monitor disease progression, target engagement and efficacy in Huntington’s disease (HD). Cerebrospinal fluid (CSF) is an ideal medium to research such biomarkers due to its proximity to the brain. Objective: To investigate the safety and feasibility of research lumbar punctures (LP) in HD. Methods: HDClarity is an ongoing international biofluid collection initiative built on the Enroll-HD platform, where clinical assessments are recorded. It aims to recruit 1,200 participants. Biosamples are collected following an overnight fast: blood via venipuncture and CSF via LP. Participants are healthy controls and HD gene expansion carriers across the disease spectrum. We report on monitored data from February 2016 to September 2019. Results: Of 448 participants screened, 398 underwent at least 1 sampling visit, of which 98.24% were successful (i.e., CSF was collected), amounting to 10,610 mL of CSF and 8,200 mL of plasma. In the total 572 sampling visits, adverse events were reported in 24.13%, and headaches of any kind and post-LP headaches in 14.86% and 12.24%, respectively. Frequencies were less in manifest HD; gender, age, body mass index and disease burden score were not associated with the occurrence of the events in gene expansion carriers. Headaches and back pain were the most frequent adverse events. Conclusion: HDClarity is the largest CSF collection initiative to support scientific research into HD and is now stablished as a leading resource for HD research. Our data confirm that research LP in HD are feasible and acceptable to the community, and have a manageable safety profile

A multi-voiced account of family entrepreneuring research: expanding the agenda of family entrepreneurship

Purpose This conceptual, multi-voiced paper aims to collectively explore and theorize family entrepreneuring, which is a research stream dedicated to investigating the emergence and becoming of entrepreneurial phenomena in business families and family firms. Design/methodology/approach Because of the novelty of this research stream, the authors asked 20 scholars in entrepreneurship and family business to reflect on topics, methods and issues that should be addressed to move this field forward. Findings Authors highlight key challenges and point to new research directions for understanding family entrepreneuring in relation to issues such as agency, processualism and context. Originality/value This study offers a compilation of multiple perspectives and leverage recent developments in the fields of entrepreneurship and family business to advance research on family entrepreneuring

Genetic risk and a primary role for cell-mediated immune mechanisms in multiple sclerosis.

Multiple sclerosis is a common disease of the central nervous system in which the interplay between inflammatory and neurodegenerative processes typically results in intermittent neurological disturbance followed by progressive accumulation of disability. Epidemiological studies have shown that genetic factors are primarily responsible for the substantially increased frequency of the disease seen in the relatives of affected individuals, and systematic attempts to identify linkage in multiplex families have confirmed that variation within the major histocompatibility complex (MHC) exerts the greatest individual effect on risk. Modestly powered genome-wide association studies (GWAS) have enabled more than 20 additional risk loci to be identified and have shown that multiple variants exerting modest individual effects have a key role in disease susceptibility. Most of the genetic architecture underlying susceptibility to the disease remains to be defined and is anticipated to require the analysis of sample sizes that are beyond the numbers currently available to individual research groups. In a collaborative GWAS involving 9,772 cases of European descent collected by 23 research groups working in 15 different countries, we have replicated almost all of the previously suggested associations and identified at least a further 29 novel susceptibility loci. Within the MHC we have refined the identity of the HLA-DRB1 risk alleles and confirmed that variation in the HLA-A gene underlies the independent protective effect attributable to the class I region. Immunologically relevant genes are significantly overrepresented among those mapping close to the identified loci and particularly implicate T-helper-cell differentiation in the pathogenesis of multiple sclerosis

The DECIDE project: from surveillance data to decision-support for farmers and veterinarians

Farmers, veterinarians and other animal health managers in the livestock sector are currently missing sufficient information on prevalence and burden of contagious endemic animal diseases. They need adequate tools for risk assessment and prioritization of control measures for these diseases. The DECIDE project develops data-driven decision-support tools, which present (i) robust and early signals of disease emergence and options for diagnostic confirmation; and (ii) options for controlling the disease along with their implications in terms of disease spread, economic burden and animal welfare. DECIDE focuses on respiratory and gastro-intestinal syndromes in the three most important terrestrial livestock species (pigs, poultry, cattle) and on reduced growth and mortality in two of the most important aquaculture species (salmon and trout). For each of these, we (i) identify the stakeholder needs; (ii) determine the burden of disease and costs of control measures; (iii) develop data sharing frameworks based on federated data access and meta-information sharing; (iv) build multivariate and multi-level models for creating early warning systems; and (v) rank interventions based on multiple criteria. Together, all of this forms decision-support tools to be integrated in existing farm management systems wherever possible and to be evaluated in several pilot implementations in farms across Europe. The results of DECIDE lead to improved use of surveillance data and evidence-based decisions on disease control. Improved disease control is essential for a sustainable food chain in Europe with increased animal health and welfare and that protects human health

A multi-voiced account of family entrepreneuring research : expanding the agenda of family entrepreneurship

Purpose This conceptual, multi-voiced paper aims to collectively explore and theorize family entrepreneuring, which is a research stream dedicated to investigating the emergence and becoming of entrepreneurial phenomena in business families and family firms. Design/methodology/approach Because of the novelty of this research stream, the authors asked 20 scholars in entrepreneurship and family business to reflect on topics, methods and issues that should be addressed to move this field forward. Findings Authors highlight key challenges and point to new research directions for understanding family entrepreneuring in relation to issues such as agency, processualism and context. Originality/value This study offers a compilation of multiple perspectives and leverage recent developments in the fields of entrepreneurship and family business to advance research on family entrepreneuring

Multiorgan MRI findings after hospitalisation with COVID-19 in the UK (C-MORE): a prospective, multicentre, observational cohort study

Introduction: The multiorgan impact of moderate to severe coronavirus infections in the post-acute phase is still poorly understood. We aimed to evaluate the excess burden of multiorgan abnormalities after hospitalisation with COVID-19, evaluate their determinants, and explore associations with patient-related outcome measures. Methods: In a prospective, UK-wide, multicentre MRI follow-up study (C-MORE), adults (aged ≥18 years) discharged from hospital following COVID-19 who were included in Tier 2 of the Post-hospitalisation COVID-19 study (PHOSP-COVID) and contemporary controls with no evidence of previous COVID-19 (SARS-CoV-2 nucleocapsid antibody negative) underwent multiorgan MRI (lungs, heart, brain, liver, and kidneys) with quantitative and qualitative assessment of images and clinical adjudication when relevant. Individuals with end-stage renal failure or contraindications to MRI were excluded. Participants also underwent detailed recording of symptoms, and physiological and biochemical tests. The primary outcome was the excess burden of multiorgan abnormalities (two or more organs) relative to controls, with further adjustments for potential confounders. The C-MORE study is ongoing and is registered with ClinicalTrials.gov, NCT04510025. Findings: Of 2710 participants in Tier 2 of PHOSP-COVID, 531 were recruited across 13 UK-wide C-MORE sites. After exclusions, 259 C-MORE patients (mean age 57 years [SD 12]; 158 [61%] male and 101 [39%] female) who were discharged from hospital with PCR-confirmed or clinically diagnosed COVID-19 between March 1, 2020, and Nov 1, 2021, and 52 non-COVID-19 controls from the community (mean age 49 years [SD 14]; 30 [58%] male and 22 [42%] female) were included in the analysis. Patients were assessed at a median of 5·0 months (IQR 4·2–6·3) after hospital discharge. Compared with non-COVID-19 controls, patients were older, living with more obesity, and had more comorbidities. Multiorgan abnormalities on MRI were more frequent in patients than in controls (157 [61%] of 259 vs 14 [27%] of 52; p<0·0001) and independently associated with COVID-19 status (odds ratio [OR] 2·9 [95% CI 1·5–5·8]; padjusted=0·0023) after adjusting for relevant confounders. Compared with controls, patients were more likely to have MRI evidence of lung abnormalities (p=0·0001; parenchymal abnormalities), brain abnormalities (p<0·0001; more white matter hyperintensities and regional brain volume reduction), and kidney abnormalities (p=0·014; lower medullary T1 and loss of corticomedullary differentiation), whereas cardiac and liver MRI abnormalities were similar between patients and controls. Patients with multiorgan abnormalities were older (difference in mean age 7 years [95% CI 4–10]; mean age of 59·8 years [SD 11·7] with multiorgan abnormalities vs mean age of 52·8 years [11·9] without multiorgan abnormalities; p<0·0001), more likely to have three or more comorbidities (OR 2·47 [1·32–4·82]; padjusted=0·0059), and more likely to have a more severe acute infection (acute CRP >5mg/L, OR 3·55 [1·23–11·88]; padjusted=0·025) than those without multiorgan abnormalities. Presence of lung MRI abnormalities was associated with a two-fold higher risk of chest tightness, and multiorgan MRI abnormalities were associated with severe and very severe persistent physical and mental health impairment (PHOSP-COVID symptom clusters) after hospitalisation. Interpretation: After hospitalisation for COVID-19, people are at risk of multiorgan abnormalities in the medium term. Our findings emphasise the need for proactive multidisciplinary care pathways, with the potential for imaging to guide surveillance frequency and therapeutic stratification