Lentiviral Vector Bioprocessing

Lentiviral vectors (LVs) are potent tools for the delivery of genes of interest into mammalian cells and are now commonly utilised within the growing field of cell and gene therapy for the treatment of monogenic diseases and adoptive therapies such as chimeric antigen T-cell (CAR-T) therapy. This is a comprehensive review of the individual bioprocess operations employed in LV production. We highlight the role of envelope proteins in vector design as well as their impact on the bioprocessing of lentiviral vectors. An overview of the current state of these operations provides opportunities for bioprocess discovery and improvement with emphasis on the considerations for optimal and scalable processing of LV during development and clinical production. Upstream culture for LV generation is described with comparisons on the different transfection methods and various bioreactors for suspension and adherent producer cell cultivation. The purification of LV is examined, evaluating different sequences of downstream process operations for both small- and large-scale production requirements. For scalable operations, a key focus is the development in chromatographic purification in addition to an in-depth examination of the application of tangential flow filtration. A summary of vector quantification and characterisation assays is also presented. Finally, the assessment of the whole bioprocess for LV production is discussed to benefit from the broader understanding of potential interactions of the different process options. This review is aimed to assist in the achievement of high quality, high concentration lentiviral vectors from robust and scalable processes

The synthesis of potential steroid receptor antagonists

The first section of this thesis presents a review of the literature on endocrine hormones, synthetic steroids, and synthetic steroid receptor antagonists. Particular emphasis is placed upon the estrogen, progesterone and glucocorticoid activity of these compounds. The structure, activity and preparation of some progesterone/glucocorticoid receptor antagonists is also reviewed. The second section of this thesis presents a route for the synthesis of 19-aryl substituted androstanes. These compounds have been designed to explore their potential antiprogestational and antiglucocorticoid activity. It is demonstrated that a bulky aromatic functionality may be introduced at the sterically hindered C-19 methyl group, by the nucleophilic attack of an organometallic nucleophile on a 10~-formyl-19-norandrostane, which is suitably protected at positions C-3 and C-17. The subsequent careful manipulation of the C-3 and C-17 functionalities has led to a succesful 11 step synthesis (scheme 4.19) of 17~-hydroxy-17a-(prop-l-yne)-19-(p-N,N- dimethylaminophenyl)androst-5-en-3-one (3.1), which should be of interest for biological evaluation

Ultra scale-down concepts to address early stage process development challenges in integrated continuous bioprocessing

The benefits of continuous bioprocessing, e.g. accelerated process development and scale-up, reduced capital costs, and standardisation, could be achieved through facility automation, universal process architecture, and the alignment of operational structures for the development and manufacturing organisations [1]. Both control strategy and rational design of universal process architecture demand an understanding of the limits and interdependence of these unit operations, and knowledge on how these could be controlled to sustain desired product quality over long periods of time. For example, to effectively implement global process control, which coordinate feed flowrates, will require information as to the impact on product quality and operational efficiency of the range of flowrates on individual process equipment. One of the advantages of continuous processing is the potential for operating plants to serve clinical development by shortening plant operation. Could the same be used in early stage process development? How does this scale match process development goals which, apart from producing material to demonstrate feasibility of the process, have broader goals such as generating envelopes of performance and experimental data for process understanding? This presentation will initiate discussion on early stage bioprocess development needs when facilities are running integrated continuous processes and envisage how the process of technology transfer from development scale to operating scale might look. We will provide insights into the challenges encountered in designing scale-down mimics of continuous unit operations such as tangential flow filtration or TFF [2] and will illustrate ultra scale-down concepts [3] which could be used to understand unit operations within a continuous platform. TFF is a key unit operation that has been cited as having potential for upstream cell separation or clarification. In a previous work [2], we successfully demonstrated a microscale TFF platform which mimicked a typical bench-scale TFF, Pellicon 2TM (Merck Millipore) based on operating conditions. We obtained similar fluxes, transmissions of antibody fragments, total protein and DNA (unpublished). This was achieved with membrane area that is smaller by 100-fold and reduced feed material by at least 10-fold. We identified that fluid transfer is a key limitation in the reduction of feed since pump requirements for continuous flow dictate the minimum volume of material needed to run the equipment efficiently. Without compatible fluid transfer technologies, material requirement for scale-down, continuous equipment will still be in the litre-scale per experiment. For investigative studies, important to identify key process parameters and quality attributes, these amounts would be prohibitive and would require more resources and time. This highlights the need to re-consider the typical use of geometrical scale-down models to evaluate continuous unit operations and requires more thought on early stage development. Otherwise, we may only be moving the cost and risk of biomanufacturing from industrial scale to the bench scale of process development. The USD approach endeavors to understand the complex engineering environment within an individual unit operation by identifying key engineering parameters and determining the critical flow regime. This insight is then developed into USD technologies and techniques to mimic larger scale operation. The approach suits the requirements during the early stages of product development when the amount of material is scarce and information about the product or process is limited. First applied to continuous centrifugation with the USD centrifugation technique, the USD concept has been extended for other unit operations. The USD techniques were powerful in revealing process interactions. They facilitate Quality by Design and help define process control strategy by determining and quantifying critical processing parameters which control the critical process attributes. References: (1) Konstantinov KB, Cooney CL. White Paper on Continuous Bioprocessing. May 20–21, 2014 Continuous Manufacturing Symposium. Journal of Pharmaceutical Sciences. 2015;104(3):813-20; (2) Rayat ACME, Lye GJ, Micheletti M. A novel microscale crossflow device for the rapid evaluation of microfiltration processes. Journal of Membrane Science. 2014;452(0):284-93; (3) Rayat ACME, Chatel A, Hoare M, Lye GJ. Ultra scale-down approaches to enhance the creation of bioprocesses at scale: impacts of process shear stress and early recovery stages. Current Opinion in Chemical Engineering. 2016;14:150-7

Ultra scale-down approaches to enhance the creation of bioprocesses at scale: impacts of process shear stress and early recovery stages

The sensitivity of biological materials to shear stress conditions encountered during large-scale bioprocessing makes successful scale up from the bench challenging. Ultra scale-down technologies seek to use just millilitre quantities to enhance our understanding of the impact of the process environment as a basis for process optimisation. They can help speed translation of new biological discoveries to market and reduce risks encountered in scale up. They are important both as process discovery tools and as preparative tools to yield material for study of subsequent stages. In this review the focus is on the early recovery stages post fermentation or cell culture and in particular the use of continuous-flow and dead-end centrifugation integrated with preparative stages (e.g. flocculation) and subsequent depth filtration. Examples range from therapeutic antibodies, to rationally engineered (synthetic biology) host strains, to stem cells for therapy

A scale-down model to investigate cell retention for continuous monoclonal antibody manufacture

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The role of membrane chemistry in Lentiviral vector clarification recovery for cell and gene therapies

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The feasibility of introducing an adult safeguarding measure for inclusion in the Adult Social Care Outcomes Framework (ASCOF):findings from a pilot study

Adult Social Care Safeguarding Survey Interview Schedule – adult at risk. (DOCX 368 kb

Immune Response Associated with Islet Xenotransplantation in Small and Large Animal Models

This chapter will review studies that examine the immune response to porcine neonatal pancreatic cell clusters (NPCC) in small and large animal models; specifically, the immune mechanisms that lead to the rejection of transplanted islet cells in mice, nonhuman primates, and humans will be discussed. In addition, current research on the in vitro and in vivo human immune responses to porcine NPCC is also included. Research into the immune responses that lead to islet cell death posttransplant allows for further understanding of how to better protect transplanted porcine NPCC in humans. Furthermore, this chapter will examine immune‐related strategies that have shown to extend the life and/or function of porcine NPCC in vitro and in vivo, including techniques that work to modulate the immune system of the islet cell donor and/or the recipient. Finally, this chapter will identify future areas of research that have yet to be examined extensively in the literature, mostly pertaining to the human immune response to porcine NPCC in the clinical setting