Gene Editing/Gene Therapies: CHALLENGE TO DEVELOP GMP COMPLIANT MANUFACTURING PROCESS FOR COXSACKIE VIRUS TYPE B3

Background & Aim: Gene therapies has become recognized for its remarkable clinical benefits in a variety of medical applications, in particular recent approval of an Ad vector-based COVID-19 vaccines have attracted recent global attention. Here, we present key considerations for GMP compliant process development for Coxsackie virus type B3 (CVB3), an oncolytic virus designed for clinical trial in triple-negative breast cancer. Methods, Results & Conclusion(s): CVB3 is a non-enveloped, linear single-strand RNA virus with a size of approximately 27-33 um in diameter. From the initial type using the zonal rotor centrifuge to the advanced type using the tangential flow filtration system and ion chromatograph, we considered the points of the design concept in constructing the manufacturing process. The final design system is constructed as a closed and single-use manufacturing system in which all processes from upstream large-scale cell culture to downstream target purification and concentration steps. In brief, HEK293 cell suspension extended in 3L serum-free medium infected with CVB3, up to 3.6 times 10 to 7 of TCID50 /mL before going to downstream steps, made total 150 mL of final products as 8.43 times 10 to 7 of TCID50/mL concentration. Although further quality control challenges remain that is removal of product-related impurities such as human cellular proteins and residual DNA/RNA to increase virus purity, this concept is effectively applicable even for other types of viruses as GMP manufacturing processes, and would be also important for technology transfer to future commercial production.Copyright © 2023 International Society for Cell & Gene Therapy

Fractional order epidemiological model of SARS-CoV-2 dynamism involving Alzheimer's disease

In this paper, we study a Caputo-Fabrizio fractional order epidemiological model for the transmission dynamism of the severe acute respiratory syndrome coronavirus 2 pandemic and its relationship with Alzheimer's disease. Alzheimer's disease is incorporated into the model by evaluating its relevance to the quarantine strategy. We use functional techniques to demonstrate the proposed model stability under the Ulam-Hyres condition. The Adams-Bashforth method is used to determine the numerical solution for our proposed model. According to our numerical results, we notice that an increase in the quarantine parameter has minimal effect on the Alzheimer's disease compartment.Copyright © 2022 The Author(s)

Comparison of vector elements and process conditions in transient and stable suspension HEK293 platforms using SARS-CoV-2 receptor binding domain as a model protein.

BACKGROUND: Mammalian cell lines are frequently used as protein expression hosts because of their ability to correctly fold and assemble complex proteins, produce them at high titers, and confer post-translational modifications (PTMs) critical to proper function. Increasing demand for proteins with human-like PTMs, particularly viral proteins and vectors, have made human embryonic kidney 293 (HEK293) cells an increasingly popular host. The need to engineer more productive HEK293 platforms and the ongoing nature of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic presented an opportunity to study strategies to improve viral protein expression in transient and stable HEK293 platforms. RESULTS: Initial process development was done at 24 deep well plate (DWP) -scale to screen transient processes and stable clonal cell lines for recombinant SARS-CoV-2 receptor binding domain (rRBD) titer. Nine DNA vectors that drove rRBD production under different promoters and optionally contained Epstein-Barr virus (EBV) elements to promote episomal expression were screened for transient rRBD production at 37 °C or 32 °C. Use of the cytomegalovirus (CMV) promoter to drive expression at 32 °C led to the highest transient protein titers, but inclusion of episomal expression elements did not augment titer. In parallel, four clonal cell lines with titers higher than that of the selected stable pool were identified in a batch screen. Flask-scale transient transfection and stable fed-batch processes were then established that produced rRBD up to 100 mg/L and 140 mg/L, respectively. While a bio-layer interferometry (BLI) assay was crucial for efficiently screening DWP batch titers, an enzyme-linked immunosorbent assay (ELISA) was used to compare titers from the flask-scale batches due to varying matrix effects from different cell culture media compositions. CONCLUSION: Comparing yields from the flask-scale batches revealed that stable fed-batch cultures produced up to 2.1x more rRBD than transient processes. The stable cell lines developed in this work are the first reported clonal, HEK293-derived rRBD producers and have titers up to 140 mg/L. As stable production platforms are more economically favorable for long-term protein production at large scales, investigation of strategies to increase the efficiency of high-titer stable cell line generation in Expi293F or other HEK293 hosts is warranted.

Process development of a SARS-CoV-2 nanoparticle vaccine.

One of the outcomes from the global COVID-19 pandemic caused by SARS-CoV-2 has been an acceleration of development timelines to provide treatments in a timely manner. For example, it has recently been demonstrated that the development of monoclonal antibody therapeutics from vector construction to IND submission can be achieved in five to six months rather than the traditional ten-to-twelve-month timeline using CHO cells [1], [2]. This timeline is predicated on leveraging existing, robust platforms for upstream and downstream processes, analytical methods, and formulation. These platforms also reduce; the requirement for ancillary studies such as cell line stability, or long-term product stability studies. Timeline duration was further reduced by employing a transient cell line for early material supply and using a stable cell pool to manufacture toxicology study materials. The development of non-antibody biologics utilizing traditional biomanufacturing processes in CHO cells within a similar timeline presents additional challenges, such as the lack of platform processes and additional analytical assay development. In this manuscript, we describe the rapid development of a robust and reproducible process for a two-component self-assembling protein nanoparticle vaccine for SARS-CoV-2. Our work has demonstrated a successful academia-industry partnership model that responded to the COVID-19 global pandemic quickly and efficiently and could improve our preparedness for future pandemic threats.

An economical and practical procedure of favipiravir synthesis for the treatment of Covid-19.

Favipiravir is a wide-spectrum antiviral generic drug that has received large attention during the recent COVID-19 pandemic. While there are synthetic strategies for favipiravir synthesis, economical procedures could contribute to industrial scale synthesis and availability. Accordingly, our efforts focused on an economic and scalable procedure for favipiravir synthesis via the 3,6-dichloropyrazine-2-carbonitrile intermediate obtained from 3-aminopyrazine-2-carboxylic acid. The process afforded favipiravir with 43% yield (from 3,6-dichloropyrazine-2-carbonitrile, by fluorination, hydroxylation, and nitrile hydrolysis reactions) and greater than 99% purity without a chromatographic purification step. Supplementary Information: The online version contains supplementary material available at 10.1007/s11696-022-02595-1.

Establishing PCR testing in Nepal for COVID-19: Challenges and opportunities

Polymerase chain reaction (PCR) invented by Kary Mullis (1983), has become the centrepiece of molecular detection of various infectious diseases including coronavirus disease 2019 (COVID-19). Many developing countries like Nepal faces various challenges and grab many future opportunities during and after establishment of molecular PCR laboratories throughout the country. This viewpoint describes the involvement of laboratory employees, development and adoption of new protocols or framework, deliberate partnership with national and international community is very efficient for the establishment of PCR laboratories. Beside this, continued alliance and nation leadership is crucial to generate a unified and sustainable PCR laboratory network in the country like Nepal. In future the established PCR laboratories can be utilized for the diagnosis of others pandemic diseases and can be used for multipurpose like in verification of infectious diseases;Oncology;Blood test;Genetic testing. Copyright © 2020, Kathmandu University. All rights reserved.

Educating During a Pandemic: Novel Educational Training for Incident Reporting via eLearning in the Radiation Therapy Department

Aim: Since 2018, steps have been undertaken to improve quality culture in the Radiation Therapy program including implementation of a new incident reporting software designed to improve ease of reporting for frontline staff. Recognizing that delivering education on incident reporting relies heavily on examples and clarification of details, an education plan involving in-person, small group sessions with interactive learning opportunities was designed. When COVID restrictions limited group gatherings, alternative delivery methods were explored with online learning being the most viable option. Our aim was to create an eLearning module that was engaging, informative, interactive, and highly example-based to meet the same learning objectives as traditional in-person education. Process: Development began with the creation of learning objectives and content developed for the person session was converted into logically ordered sections with clinical application examples and check-in activities for the online environment. Topical outline included a review of terminology, why and what to report, and how to use the software. Interactive activities reinforced knowledge and provided practice in identifying what should be reported and in critiquing documentation. The final product was created using Articulate360 authoring tool. As a part of the eLearning course, a pre and post self-assessment survey was embedded to measure the perceived effectiveness of the course contents. Benefits/Challenges: Creating educational content in an eLearning module provides increased accessibility for learners, permits ad hoc refreshers as needed, fosters consistency in disseminated information, and is potentially a starting point for a catalogue of courses available for onboarding new staff. Challenges included limiting information per adult learning principles, editing content to ensure visual appeal, and resource demands on instructional designers. Due to an increase in hospital-wide requests for eLearning modules in response to COVID restrictions, there was a significant wait time for initiation of the project. Impact/Outcomes: Based on the results of the pre and post-assessment surveys, 95% of learners reported that they had an increase in understanding of both incident reporting and Improvement Flow. This is a considerably favorable outcome and is indicative that we have successfully implemented an eLearning module that is engaging and informative and that meets the learning objectives originally intended for in-person education. Self-assessment regarding one's abilities to know when to report an incident that: causes patient harm (medical or dosimetric), reaches the patient but does NOT cause harm and that is caught and corrected before reaching the patient each increased by 17.5% after completion of the course with an overall positive response rate of 95%, 100%, and 97.5% respectively. In regards to self-assessment of one's abilities to use Improvement Flow, results indicated a positive 32.5% increase post-module completion with 95% of respondents indicating they are comfortable using the program. An additional 32.5% improvement was seen in response to individuals' self-assessment of their abilities to recognize when a situation is reportable with an overall 95% of respondents indicating positive assessments. Our experience indicates that training that requires interactive, example-based learning and clarification of details is capable of being delivered via an eLearning platform with robustly positive results. Copyright © 2022

Development and Co-Design of a Digital Intervention for People Affected by Dementia who are Fearful of Disclosing the Diagnosis to Their Social Networks

Background: Receiving a diagnosis of dementia can affect all aspects of an individual's life, including their sense of identity. As a consequence, some people with dementia may socially withdraw due to stigma, embarrassment or fear of what might happen if other people were to find out about the diagnosis. The "Who to tell, how and when" intervention, a group-based face-to-face intervention, has been developed for people affected by dementia who are fearful of disclosing the diagnosis to others. This study aims to adapt the existing intervention for online delivery, in light of the ongoing COVID-19 pandemic. Method(s): A co-created, user-centred design in three phases is applied. In phase 1, focus groups conducted with people with dementia and informal caregivers will inform the platform, features and adaptation of pre-existing material. Phase 2 consists of the development of the digital prototype. In phase 3, the feasibility and usability are tested with end users to inform prototype v. 2.0. Result(s): Research has indicated that peer support in the disclosure decision-making process is especially important for participants. Results from phase 1 and 2, including findings from the discussions with the focus groups and options appraisals for the peer support elements of the digital intervention will be discussed. We will discuss general principles of adaptation from paper manual to digital intervention. Conclusion(s): Online access to this intervention may be especially valuable for people who wish to maintain anonymity and privacy, for example people from cultural or ethnic groups where the stigma of dementia is high. Online versions of face-to-face interventions improve overall accessibility by providing choice. Copyright © 2022 the Alzheimer's Association.

Process Development and Manufacturing: THE IMPACT OF THE COVID-19 PANDEMIC ON THE CELL AND GENE THERAPY SUPPLY CHAIN

Background & Aim: The SARS-CoV-2 (COVID-19) pandemic caused global challenges throughout 2020. Cell and Gene Therapy (CGT) trials were halted initially to allow healthcare workers and supply manufacturers to address the immediate and urgent needs from the pandemic. The initial decrease in cases of the alpha variant led to the re-launch of many of these trials;however, critical supplies required for manufacturing and administration of products have continued to be in short supply or available with prolonged lead times. An initial survey for CGT Critical Supply Challenges was distributed to the field in late October 2021 and open for two weeks to obtain a baseline understanding of the key challenges. This survey was distributed a second time in early 2022 to assess the changes in critical supply chain shortages over the intervening months. Here we report the cumulative results of the two surveys and describe the continued impact of the COVID-19 pandemic on the critical supply chain within the Cell and Gene Therapy field. Methods, Results & Conclusion: Methods. The survey was created using SurveyMonkey®, an online platform, and contained 10 questions aimed to understand key supply shortages in the context of product type and stage of production. The survey was disseminated through CGT society networks, Google Groups (Small Cell Therapy Lab), LinkedIn®, and personal emails. The survey was distributed initially for one week in October 2021 and data was analyzed. The survey (Figure Presented) was distributed a second time in early 2022 and results from the second survey were analyzed separately and compared to the initial results. Results. The initial survey contained 58 responses and 91% of these had experienced a critical supply shortage in the past 18 months. The most common supply category causing the largest impact was transfer supplies, including plastics, tubing, bags and vials (Fig 1). Half of the respondents experienced a backorder of over 6 months (Fig 2);however, 40% of respondents did not have to delay productions (Fig 3). Supply shortages have continued into 2022 and the second distribution of the survey is to assess any changes in types of supplies that are delayed. Conclusion. A majority of survey respondents have dealt with supply chain challenges since the onset of the COVID-19 pandemic. Shortages and long lead times continue to be present throughout early 2022;however, groups have been able to continue productions through a variety of mechanisms. (Figure Presented) (Figure Presented)

Process Development and Manufacturing: EXPANSION OF DIFFERENT TYPES OF THERAPEUTIC CELLS USING XENOGENEIC FREE AND GAMMA IRRADIATED HUMAN PLATELET LYSATE

Background & Aim: Mill Creek’s human platelet lysate (hPL) is produced using expired human platelets obtained from accredited blood banks in the United States. These platelets were originally intended for use in patient transfusion. The safety of platelets used in transfusion is managed by the U.S. Food & Drug Administration (FDA), as well as the American Association of Blood Banks (AABB). These organizations set standards, including testing for transmissible diseases. The United States record for blood safety is well established, with extremely low rates of disease transmission, making the platelet units used for hPL manufacture low risk. However, over the past few decades, the number of emerging infectious diseases that spread to people has increased. Even though transmission of Covid-19 and other potential emergent pathogens via blood transfusion has not been documented, adventitious agents remain a concern. For that reason, pathogen reduction processes are necessary to improve the safety profile of blood-derived products used in the manufacture of cellbased therapies. One of these processes is the pathogen reduction through gamma irradiation. Methods, Results & Conclusion: Previously, we validated a process to produce gamma irradiated human platelet lysate. To be able to release the gamma irradiated product as a pathogen reduced product, we additionally performed a viral clearance study, which determined the efficacy of the process to actually reduce a potential viral contaminant. Here, we present the results obtained from the viral clearance study performed on our gamma irradiated hPL, and from the expansion of different types of therapeutic cells using our gamma irradiated hPLs, which showed unprecedented levels of product quality and efficiency. Our products are a game changer for the growing market of cell base therapies bioprocessing and manufacturing.

Process Development and Manufacturing: A SUPPLY CHAIN CRISIS STORY: CULTURE BAG SHORTAGE ENFORCED VALIDATION OF AN ALTERNATIVE EXPANSION SYSTEM FOR CAR T CELLS

Background & Aim: The recent supply chain crisis highlights a need to establish alternative manufacturing (MFG) protocols ensuring continuity of existing and new cell therapy (CT) clinical trials. Our academic CT program, and likely others, experienced purchasing delays and restrictions caused by diversion of critical supplies to meet COVID-19- related research demands and/or reduced vendor capacity due to resource constraints, including attrition of skilled workforce. Mitigation strategies aimed at creating process redundancies overcome production challenges resulting from a scarcity of goods. Here, we validated an alternative ex vivo culture system to clinically MFG lentiviral vector (LV) modified CAR T cells due to limited availability of cell expansion culture bags for the Wave bioreactor, a critical unit of operation that we have used to successfully MFG thousands of gene-modified T cell products for 30+ clinical trials. Methods, Results & Conclusion: The disposable G-REX culture vessels were compatible and seamlessly integrated with our closed system platform. Mesothelin CAR T cells were manufactured in parallel via the G-REX or conventional Wave bioreactor using consented patient starting material. Critical quality attributes of the final T cell products, including viability, transduction efficiency, phenotype and function were assessed. Transduction efficiencies assessed by flow cytometry and/or molecular qPCR were lower in products generated in the G-REX compared to the wave using the same multiplicity of infection. However, at least 50-fold expansion was achieved, with cell viabilities greater than 90% and with comparable cellular phenotypes. The Meso CAR T cells generated by either process were capable of eliciting CAR-mediated cytotoxicity and effector cytokine production. Strikingly, 2-4 billion T cells were harvested from a starting seed number of just 50 million T cells in the 1L G-REX, which may be sufficient to meet most protocol- specified cell therapy doses, suggesting that a full apheresis collection may not be needed. Notably, this process required just 1/3 of the starting material, 1/5 of the media and decreased manual effort through culture duration compared to the Wave. Additionally, the reduced reliance on specialized capital equipment combined with a small footprint enables simultaneous MFG of several immunotherapy products. These advantages propose consideration in replacement of current expansion platform as well as validating an alternative process for MFG CAR T cells.

Digital transformation in practice based learning

Keywords: Digital;Placement;Impact Purpose: Since February 2020 COVID 19 has presented significant challenges to physiotherapy staff and had devastating impact on many services putting continued provision of practice based learning (PBL) at significant risk. Academic and clinical professionals have been compelled to develop safe alternatives to traditional PBL thereby ensuring students have adequate opportunity to meet statutory and professional body requirements. The NHS Long Term Plan (NHS, 2019a) and Interim People plan (NHS, 2019b identify digital transformation and significant growth of the Allied Health Profession workforce as essential for the continued delivery of high quality health and social care. Implementation of these plans is contingent on both adequate volume and a continuous pipeline of digitally able AHP graduates. Working in partnership, Teesside University and Tees Esk & Wear Valley NHS Foundation Trust coproduced a technology enhanced 5 week service development and leadership learning opportunity within the clinical specialisms of Mental Health and Learning Disability to enable continued high quality PBL. Methods: An iterative process of development began with wide ranging consultation and discussion across organisations and professional networks to inform the structure and content of the 5 week experience. A programme of learning was developed comprising: formal teaching, individual and group directed learning, consultation, individual, peer and group supervision Due to the new and novel nature of this PBL, students were briefed and invited to express interest in involvement;six students were subsequently allocated. In addition to standard evaluation processes all students took part in a focus group to enable them to share reflections and give comprehensive feedback to academic and clinical staff. All clinical staff completed a feedback questionnaire to inform future development. Results: Six students successfully completed 5 weeks technology enhanced PBL. All reported that this opportunity enabled them to achieve their personal, clinical, professional and statutory body outcomes and standards with a rounded experience of Mental Health and Learning Disabilities services. Students felt focused 1:1, group and peer supervision provided enhanced support and enabled high levels of learning and achievement. Students, clinical and academic staff all suggested the opportunity should continue with only minor modification to the current format. Placement capacity significantly increased. Whilst initial organisation was time consuming, with an overarching framework in place this will be considerably reduced for future occurrences. Conclusion(s): The revered model of PBL which is patient facing and involves a 1:1 model of supervision can be effectively replaced with technology enhanced non patient facing experience using an innovative mixed methods model of supervision. This approach can provide students with insight and experience which is not possible via traditional PBL, develop their digital literacy, confidence, resilience and emotional intelligence and foster a broad understanding of healthcare. Impact: This framework can be translated across organisations, settings and specialties offering solutions to the challenges presented by COVID19 and offer a high quality solution to ongoing PBL capacity deficit whilst adding value to the experience and development of students and clinical staff. Funding acknowledgements: No funding was sourced/required.

Vero cell upstream bioprocess development for the production of viral vectors and vaccines.

The Vero cell line is considered the most used continuous cell line for the production of viral vectors and vaccines. Historically, it is the first cell line that was approved by the WHO for the production of human vaccines. Comprehensive experimental data on the production of many viruses using the Vero cell line can be found in the literature. However, the vast majority of these processes is relying on the microcarrier technology. While this system is established for the large-scale manufacturing of viral vaccine, it is still quite complex and labor intensive. Moreover, scale-up remains difficult and is limited by the surface area given by the carriers. To overcome these and other drawbacks and to establish more efficient manufacturing processes, it is a priority to further develop the Vero cell platform by applying novel bioprocess technologies. Especially in times like the current COVID-19 pandemic, advanced and scalable platform technologies could provide more efficient and cost-effective solutions to meet the global vaccine demand. Herein, we review the prevailing literature on Vero cell bioprocess development for the production of viral vectors and vaccines with the aim to assess the recent advances in bioprocess development. We critically underline the need for further research activities and describe bottlenecks to improve the Vero cell platform by taking advantage of recent developments in the cell culture engineering field.