Reducing risks with a serum-free medium for MRC-5 based vaccine production

The World Health Organization has set limits to the amount of bovine serum albumin (BSA) in vaccines to 50ng/dose. Vaccines that are produced with MRC-5 or other diploid cells are cultured in classical medium with bovine serum. Fetal bovine serum contains on average 23g/L BSA which adds a burden to downstream vaccine formulation. To reduce risks associated with bovine sera, we have developed an animal origin-free vaccine production medium for diploid cells. The medium is paired with a serum-free growth medium that supports direct recovery from thaw and adaptation-free expansion, while resulting in performance that is comparable to serum-containing medium. We confirmed virus production with varicella zoster virus and vesicular stomatitis virus (as an analog to rabies virus) and demonstrated titers that were up to one log higher than classical medium control cultures. Taken together, we have developed a workflow for diploid cells consisting of a serum-free medium for growth and an animal origin-free medium for virus production. By switching to a serum-free process, vaccine manufacturers can reduce dependency on serum, production and purification costs, and increase product consistency and safety

Optimizing scale-up of Vero cells cultured on microcarriers in serum-free medium for vaccine production

Vaccine production with adherent cell lines faces multiple challenges which include selection of a suitable vessel, detachment of cells for scale up, optimization of infection, as well as harvest of virus particles. Microcarriers greatly increase the surface area for adherent cells and offer flexibility for expansion to bioreactors, but scale-up methods require optimization of bead-to-bead transfer. Even though the majority of cell culture based vaccines are produced with adherent cell lines, literature provides limited information in regards to optimization of adherent cell line processes. Some process improvements have been achieved; for example, recent advances in serum free media which no longer require medium exchange prior to virus infection. In this study we focus on the production of the rabies virus surrogate, vesicular stomatitis virus, in Vero cells. Using Cytodex-1 microcarriers in spinner flasks, we evaluated effects of intermittent and continuous stirring, detachment of cells, variation in the addition of new microcarriers on the growth of Vero cells, and effects on vesicular stomatitis virus production. Viable cell density measurements revealed that initial intermittent stirring resulted in increased cell densities compared to continuous stirring after microcarrier addition. In an effort to further simplify the process, we demonstrate that detachment of cells was not required to facilitate bead-to-bead transfer on Cytodex-1 microcarriers

The memory space: Exploring future uses of Web 2.0 and mobile internet through design interventions.

The QuVis Quantum Mechanics Visualization project aims to address challenges of quantum mechanics instruction through the development of interactive simulations for the learning and teaching of quantum mechanics. In this article, we describe evaluation of simulations focusing on two-level systems developed as part of the Institute of Physics Quantum Physics resources. Simulations are research-based and have been iteratively refined using student feedback in individual observation sessions and in-class trials. We give evidence that these simulations are helping students learn quantum mechanics concepts at both the introductory and advanced undergraduate level, and that students perceive simulations to be beneficial to their learning.Comment: 15 pages, 5 figures, 1 table; accepted for publication in the American Journal of Physic

Regulation of the human p21((waf1/cip1)) gene promoter via multiple binding sites for p53 and the vitamin D(3) receptor

The main regulator of the human tumor suppresser gene p21((waf1/cip1)) is the transcription factor p53, but more recently it has been suggested to be a primary anti-proliferative target for the nuclear receptor VDR in the presence of its ligand 1α,25-dihydroxyvitamin D(3) (1α,25(OH)(2)D(3)). To identify VDR responding regions, we analyzed 20 overlapping regions covering the first 7.1 kb of the p21((waf1/cip1)) promoter in MCF-7 human breast cancer cells using chromatin immuno-precipitation assays (ChIP) with antibodies against p53 and VDR. We confirmed two known p53 binding regions at approximate positions −1400 and −2300 and identified a novel site at position −4500. In addition, we found three VDR-associated promoter regions at positions −2300, −4500 and −6900, i.e. two regions showed binding for both p53 and VDR. In silico screening and in vitro binding assays using recombinant and in vitro translated proteins identified five p53 binding sites within the three p53-positive promoter regions and also five 1α,25(OH)(2)D(3) response elements within the three VDR-positive regions. Reporter gene assays confirmed the expected responsiveness of the respective promoter regions to the p53 inducer 5-fluorouracil and 1α,25(OH)(2)D(3). Moreover, re-ChIP assays confirmed the functionality of the three 1α,25(OH)(2)D(3)-reponsive promoter regions by monitoring simultaneous occupancy of VDR with the co-activator proteins CBP, SRC-1 and TRAP220. Taken together, we demonstrated that the human p21((waf1/cip1)) gene is a primary 1α,25(OH)(2)D(3)-responding gene with at least three VDR binding promoter regions, in two of which also p53 co-localizes

Improving vaccine production with a serum-free medium for MRC-5 cells

MRC-5 cells are used for vaccine production, including varicella zoster virus, MMR, polio, rotavirus, rabies, hepatitis A, and dengue virus. As human diploid fibroblast cells, MRC-5 can be passaged for about 42-48 population doublings before they become senescent. Vaccine manufacturers generally culture diploid cells in classical medium with 10% serum. Due to the limited population doublings, there is a short window for adaptation to serum-free medium and successful scale-up for vaccine production. Here, we report the development of a serum-free medium for MRC-5 cells that allows for direct adaptation and reaches comparable doubling times and virus titers as serum-containing medium. Using metabolite analysis and a design of experiment rationale, we developed a serum-free medium for growth of MRC-5 cells that can support direct recovery from thaw and adaptation-free expansion, while resulting in performance that is comparable to serum-containing medium. Since requirements for production of viruses are different from cell growth, we optimized the production medium separately. With the animal-origin-free production medium, manufacturers can produce vaccines without concern about the bovine serum albumin limit of 50ng/dose as set by the WHO. We confirmed virus production with varicella zoster virus and vesicular stomatitis virus (as an analog to rabies virus) and demonstrate titers that are up to one log higher than classical medium control. Taken together, we have developed a serum-free medium for MRC-5 cells that supports growth and virus production. By switching to a serum-free process, vaccine manufacturers can reduce dependency on serum, production and purification costs, and increase product consistency and safety

Manaakitia te paharakeke: an insight into the daily operational challenges facing Te Whakaruruhau

This report is based on participant observations of the daily operations at Te Whakaruruhau, Hamilton. The observations represent the fieldwork element of an undergraduate paper contributing to the co-author, Anna Kurei’s Bachelor’s degree at the University of Waikato. Anna’s participation and observations over several weeks included attending meetings, shadowing Advocates, contributing (as appropriate) to operations and observing interactions between Advocates and the women in living in the service’s residential housing. Founded by Ruahine Albert and Ariana Simpson in 1986, Te Whakaruruhau Incorporated (Waikato women's refuge) was the first Māori women's refuge in Aotearoa. Since its inception, Te Whakaruruhau has been a Kaupapa driven service, with Māori cultural practices consciously employed throughout all its operations. Māori tikanga is fluid and adaptable by nature and can therefore meet the needs of people from multiple cultures and backgrounds. The current service has grown from humble beginnings in a four-bedroom state owned house providing emergency housing, to now include a twenty-four-hour crisis service, residential housing and a broadened community outreach programme. Staff numbers have increased from 7 to 36 paid staff and the twenty-fourhour crisis service has allowed the refuge to provide services for high risk cases that would otherwise be turned away. Funding is critical to the successful operation of the service. The refuge provides wrap-around services to meet clients’ needs and help them navigate through a maze of government and community services. The needs of women and families who have lived with domestic violence are deep-seated and complex. Achieving a stable, healthy, independent life is frequently a long-term process. Funding however, is not only limited, but is tied to expectations of achieving successful outcomes in the short-term. It was quickly evident during the fieldwork that Te Whakaruruhau is desperately under-resourced. In the year to June 2015 the Refuge provided services for 6575 cases, but had contracted funds for less than 1600. The consequences of such starkly inadequate resources are dire – for both clients and staff. Advocates (case workers) are frequently exhausted as they try to assist women and children with high and complex needs with very little resources on a highly restricted budget. Many times, workers were observed relegating their own interests (including their own health and safety) in order to meet the demands and needs of their clients. Similarly, the successful rehabilitation of clients is jeopardised by restricted options, insufficient capacity in the system and at times even the simplest of requirements such as transport to essential services. We know that when women and children become free of violence they have better health, employment and education outcomes. These outcomes benefit not only themselves but their communities and the wider social and economic landscape. Higher levels of funding - with a longer term focus- would therefore ultimately reduce the costs of domestic violence overall. Further to this, Kaupapa-based services at Te Whakaruruhau offer a culturally meaningful response to the high representation of Maori women seeking assistance. Its success in the face of such high levels of under-resourcing suggests that expectations around funding also need to be altered

The next generation of fibroblast-based vaccine development

Chicken embryo fibroblasts (CEF) and diploid cells have a long history in vaccine production since their isolation in the 1960s at the Wistar Institute (WI-38 cells) as well as the Medical Research Council (MRC-5 cells). The cells quickly became adopted for a number of vaccines: varicella zoster (VZV), MMR, yellow fever, polio, hepatitis A, rotavirus, rabies, Marek\u27s disease, and dengue virus. Most of these vaccine processes were developed with classical media supplemented with Fetal Bovine Serum (FBS). The Hayflick limit of diploid cells restricted their adaptation to a serum-free process. While some of the vaccines such as polio and rabies have been transitioned to Vero cells, several vaccines continue to be manufactured with CEF and human diploid cells. Currently, FBS from Australia and New Zealand are utilized for the highest level of patient safety for human vaccines. However, this supply of serum is challenged by two factors: growth of existing vaccines to improve global access and the development of new gene therapies that require FBS. In order to reduce dependency on serum, we initiated a medium development program. Using metabolite analysis and DOE, we have developed a serum-reduced growth medium and a serum-free virus production medium for MRC-5 and other fibroblast cells. With a serum reduction of 90-100%, the growth medium can support direct recovery from thaw and adaptation-free expansion, resulting in performance that is comparable to classical medium with 10% serum. We confirmed virus production with VZV and vesicular stomatitis virus in MRC-5 cells as well as Marek’s disease virus in CEFs and demonstrate a higher specific productivity. By switching to a low serum process, vaccine manufacturers can reduce production and purification costs, and increase product consistency and safety

Improving estimates of growth for pearl perch (Glaucosoma scapulare) in Queensland, Australia

The pearl perch (Glaucosoma scapulare) is endemic to the east coast of Australia in depths to 150 m. The species has a long history of exploitation, and the stock is currently depleted. Previous research indicated the species is long lived and slow growing based on fishery-dependent sampling undertaken in the late 1990s and early 2000s on traditional fishing grounds at the southern end of the species’ range. Increasing fishing power has facilitated the expansion of the fishery to areas to the north and east of traditional grounds, which has resulted in the appearance of older fish (>10 yr) in fishery-dependent samples not previously observed. The current study estimated the growth parameters using 1153 length-at-age observations from fish collected in Queensland between January 2020 and December 2021. The lack of significant numbers of individuals at either end of the age frequency distribution necessitated the estimation of growth in a Bayesian framework with informative priors for length-at-age-zero and maximum length using a multi-model approach. The von Bertalanffy growth function (VBGF) was found to best fit the observed length-at-age data and the estimated VBGF parameters were L∞ = 562 mm FL, L0 = 2.02 mm FL and k = 0.295 yr−1. The high proportion of older fish in samples, combined with prior information on relevant parameters, improves growth parameter estimation by reducing bias and facilitating improved model fits to observed length-at-age data

Improving estimates of growth for pearl perch (Glaucosoma scapulare) in Queensland, Australia

Abstract The pearl perch (Glaucosoma scapulare) is endemic to the east coast of Australia in depths to 150 m. The species has a long history of exploitation, and the stock is currently depleted. Previous research indicated the species is long lived and slow growing based on fishery-dependent sampling undertaken in the late 1990s and early 2000s on traditional fishing grounds at the southern end of the species’ range. Increasing fishing power has facilitated the expansion of the fishery to areas to the north and east of traditional grounds, which has resulted in the appearance of older fish (>10 yr) in fishery-dependent samples not previously observed. The current study estimated the growth parameters using 1153 length-at-age observations from fish collected in Queensland between January 2020 and December 2021. The lack of significant numbers of individuals at either end of the age frequency distribution necessitated the estimation of growth in a Bayesian framework with informative priors for length-at-age-zero and maximum length using a multi-model approach. The von Bertalanffy growth function (VBGF) was found to best fit the observed length-at-age data and the estimated VBGF parameters were L∞ = 562 mm FL, L0 = 2.02 mm FL and k = 0.295 yr−1. The high proportion of older fish in samples, combined with prior information on relevant parameters, improves growth parameter estimation by reducing bias and facilitating improved model fits to observed length-at-age data